Chemical compounds

ABSTRACT

The present invention provides a compound of a formula (I): or a pharmaceutically acceptable salt thereof; a process for preparing such a compound; and to the use of such a compound in the treatment of an ENaC mediated disease state (such as asthma, CF or COPD).

The present invention concerns pyrazine derivatives havingpharmaceutical activity, to processes for preparing such derivatives, topharmaceutical compositions comprising such derivatives and to the useof such derivatives as active therapeutic agents.

Hydration of the lung airway epithelium ensures efficient ciliaryfunction, and mucociliary clearance (MCC) is a critical first lineairway innate defense mechanism. Failure of adequate mucus clearanceproduces mucus stasis and risk of airway obstruction and predisposes tochronic bacterial infection. Mucus hypersecretion, thickening of themucus and reduced rates of mucociliary clearance are well establishedcharacteristics of both Cystic Fibrosis (CF) and Chronic ObstructiveLung Disease (COPD) pathophysiology, and contribute significantly to themorbidity and mortality of the diseases. Poor MCC leads to airwayobstruction by static mucus, which impairs lung function and serves as anidus for infection facilitating bacterial colonization and leading toincreased rates and severity of disease exacerbations.

The pathophysiology of CF is well characterized and is caused bymutations in the cystic fibrosis transmembrane conductance regulator(CFTR) gene leading to airway dehydration, and remains one of the mostcommon fatal hereditary disorders worldwide. Although CF is a complexmulti-organ disease, morbidity and mortality are mainly determined bychronic obstructive lung disease that evolves from early onset mucusplugging in the small airways, chronic neutrophilic airway inflammationand bacterial infection.

The pathophysiology of COPD is complex and poorly understood. Currentclinical guidelines describe COPD as a disease state characterized byairflow limitation that is not fully reversible. The airflow limitationis usually both progressive and associated with an abnormal inflammatoryresponse of the lungs to noxious particles and gases. The most importantcontributory source of such particles and gases is tobacco smoke. COPDpatients have a variety of symptoms, including cough, shortness ofbreath, and excessive production of sputum; such symptoms arise fromdysfunctions of a number of cellular compartments, includingneutrophils, macrophages, and epithelial cells.

Adequate airway hydration is of key importance for maintaining MCC andappropriate water content of mucus. The regulation of airway hydrationis strongly linked to electrolyte and water movement over the epithelialcell layer. The periciliary liquid (PCL) on the airway epithelialsurface surrounding the cilia separates the viscous mucus layer from theepithelial surface and facilitates ciliary beating. The volume/depth ofthe PCL has been shown to be a strong determinant of MCC rate.

ENaC (the Epithelial Sodium Channel) is an amiloride-sensitive,non-voltage gated ion channel widely expressed in epithelia of therespiratory, urinary, digestive, genital systems and skin. Systemically,ENaC plays a key role in electrolyte homeostasis and fluid volumebalance by regulating Na⁺ transport over the kidney epithelia. Throughcontrol of sodium transport, ENaC controls the osmotic gradient over theairway epithelium and thereby regulates the PCL volume. BlockingENaC-mediated sodium transport reduces the osmotic gradient over theepithelium, leads to retention of water on the airway surface (increasedPCL volume), and enhancement of mucus hydration and MCC rate.

A role for ENaC as a key regulator of airway surface liquid (ASL) volumeand mucociliary clearance (MCC) in vivo is established in theliterature. Studies on α- β- and γ-ENaC (−/−) mice, respectively, show acritical role of ENaC function in perinatal lung liquid clearance(Barker et al., J. Clin. Invest., 1998. 102(8):1634; Bonny and Hummler,Kidney Int., 2000. 57(4):1313; Pradervand et al., Proc. Natl. Acad. Sci.USA., 1999. 96(4):1732). ENaC β-subunit overexpressing transgenic miceshow increased airway Na⁺ absorption, ASL volume depletion, mucusdehydration and delayed MCC. The mice develop severe lung disease withclinical features similar to CF and COPD, including mucus obstruction,goblet cell metaplasia, neutrophilic inflammation, defective bacterialclearance and emphysema (Mall et al., Nature Med., 2004. 10:487; Mall etal., Am. J. Respir. Crit. Care Med., 2007. 177:730). The mortality rateamong the transgenics during their first 20 days of life is around 50%due to extensive mucus plugging of the airways resulting in asphyxia,but mortality is significantly reduced by administration of amiloride tothe lungs of the mice for 14 days after birth (Mall et al., ATS Posterabstract #G55, 2008).

Furthermore, humans with Pseudohypoaldosteronism 1 (PHA1), a diseasecaused by loss-of-function mutations in the genes encoding α- β- andγ-ENaC, show increased ASL volume and an upregulation of MCC (Kerem etal., N. Engl. Med., 1999. 341:156). Treatment of normal subjects withthe ENaC channel blocking compound amiloride, increases ASL volume andMCC rates (Sood et al., Am. J. Crit. Care Med., 2003. 167:158).

Amiloride and benzamil are pharmaceutically active pyrazine derivativesknown to blockade the epithelial sodium channel.

Briefly, this specification describes, in part, a compound of formula(I):

wherein:

R¹ is selected from hydrogen or C₁₋₄ alkyl;

m is 1 or 2;

A is selected from phenyl or heterocyclyl;

X is selected from —C(═O)—, —C(═O)—NR⁴— or —O—C(═O)—NR⁵—;

n is 2 or 3;

R² is selected from hydrogen or C₁₋₈ alkyl;

R³ is C₅₋₆ alkyl-OH, wherein the said C₅₋₆ alkyl group is furthersubstituted by an additional 3 or 4 —OH groups; and

R⁴ and R⁵ are selected from hydrogen or C₁₋₄ alkyl;

or a pharmaceutically acceptable salt thereof.

This specification also describes, in part, pharmaceutical compositionswhich comprise a compound of the formula (I), or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable adjuvant,diluent or carrier.

This specification also describes, in part, a compound of formula (I),or a pharmaceutically acceptable salt thereof, for use in therapy.

This specification also describes, in part, a compound of formula (I),or a pharmaceutically acceptable salt thereof, for the treatment orprevention of an ENaC mediated disease state.

This specification also describes, in part, a compound of formula (I),or a pharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment or prevention of an ENaC mediated diseasestate.

This specification also describes, in part, a method of treating or ofpreventing an ENaC mediated disease state in a mammal suffering from, orat risk of, said disease, which comprises administering to a mammal inneed of such treatment a therapeutically effective amount of a compoundof formula (I), or a pharmaceutically acceptable salt thereof.

Further aspects of the invention will be apparent to one skilled in theart from reading this specification.

The compounds of the invention may exist in salt-form or in non-saltform (ie. as a free base), and the present invention covers both saltforms and non-salt forms.

Compounds described in this specification may form acid addition salts.In general, an acid addition salt can be prepared using variousinorganic or organic acids. Such salts can typically be formed by, forexample, mixing the compound with an acid (e.g. a stoichiometric amountof an acid) using various methods known in the art. This mixing mayoccur in water, an organic solvent (e.g. ether, ethyl acetate, ethanol,methanol, isopropanol, or acetonitrile), or an aqueous/organic mixture.

In another aspect of the invention acid addition salts are, for example,trifluoroacetate, formate, acetate or hydrochloric.

The skilled person will be aware of the general principles andtechniques of preparing pharmaceutical salts, such as those describedin, for example, Berge et al., J. Pharm. Sci., 66, 1-19 (1977).

Compounds and salts described in this specification may include one ormore chiral (i.e. asymmetric) centres. To the extent a structure orchemical name in this specification does not indicate the chirality, thestructure or name is intended to encompass any single stereoisomer (i.e.any single chiral isomer) corresponding to that structure or name, aswell as any mixture of stereoisomers (e.g. a racemate). In someembodiments, a single stereoisomer is obtained by isolating it from amixture of isomers (e.g. a racemate) using, for example, chiralchromatographic separation. In other embodiments, a single stereoisomeris obtained through direct synthesis from, for example, a chiralstarting material.

When in solid crystalline form a compound of formula (I) can be in theform of a co-crystal with another chemical entity and the inventionencompasses all such co-crystals.

The compounds of the invention may exist as a solvate (such as ahydrate) as well as unsolvated forms, and the present invention coversall such solvates.

Compounds and salts described in this specification may exist in varioustautomeric forms and the invention encompasses all such tautomericforms. “Tautomers” are structural isomers that exist in equilibriumresulting from the migration of a hydrogen atom.

Compounds and salts described in this specification may beisotopically-labeled (or “radio-labeled”). In that instance, one or moreatoms are replaced by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number typically found in nature.Examples of radionuclides that may be incorporated include ²H (alsowritten as “D” for deuterium), ³H (also written as “T” for tritium),¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O and ³⁶Cl. The radionuclide thatis used will depend on the specific application of that radio-labeledderivative. For example, for in vitro receptor labeling and competitionassays, ³H or ¹⁴C are often useful. For radio-imaging applications, ¹¹Cis often useful. In some embodiments, the radionuclide is ³H. In someembodiments, the radionuclide is ¹⁴C. In some embodiments, theradionuclide is ¹¹C.

Alkyl groups and moieties are straight or branched chain, e.g. C₁₋₈alkyl, C₁₋₆ alkyl, C₁₋₄ alkyl or C₅₋₆ alkyl. Examples of alkyl groupsare methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl,n-hexyl, n-heptyl and n-octyl, such as methyl or n-hexyl.

Heterocyclyl is a non-aromatic 5 or 6 membered ring comprising one ortwo heteroatoms independently selected from nitrogen, oxygen or sulphur;or an N-oxide thereof, or an S-oxide or S-dioxide thereof. An example ofa heterocyclyl is a non-aromatic 5 or 6 membered ring comprising one ortwo nitrogen atoms; or an N-oxide thereof, or an S-oxide or S-dioxidethereof, for example, pyrrolidinyl or piperidinyl, such aspiperidin-4-yl. For the avoidance of doubt, substituents on theheterocyclyl ring may be linked via either a carbon atom or aheteroatom.

The term “pharmaceutically acceptable” is used to characterize a moiety(e.g. a salt, dosage form or excipient) as being suitable for use in theintended patient.

In one particular aspect R¹ is substituted to the phenyl group in the2-position relative to the point of attachment to the remainder of theformula (I).

In a further aspect R¹ is C₁₋₄ alkyl.

In a further aspect R¹ is methyl.

In a further aspect m is 1.

In a still further aspect A is selected from phenyl or piperidinyl.

In a further aspect A is phenyl.

In a further aspect A is piperidinyl.

In another aspect X is —C(═O)—NR⁴—.

In a further aspect X is —C(═O)—NH—.

In one aspect, where the group X is —C(═O)—NR⁴—, then the carbonyl of Xis linked to A and the amino group of X to (CH₂)_(n). In another aspect,where the group X is —C(═O)—NR⁴—, then the amino group of X is linked toA and the carbonyl group of X to (CH₂)_(n).

In one aspect, where the group X is —O—C(═O)—NR⁵—, then the ether oxygenof X is linked to A and the amino group of X to (CH₂)_(n). In anotheraspect, where the group X is —O—C(═O)—NR⁵—, then the amino group of X islinked to A and the ether oxygen of X to (CH₂)_(n).

In another aspect the group X is substituted to the group A in the4-position relative to the point of attachment to the remainder of thecompound of formula (I).

In another aspect n is 2.

In a further aspect R² is C₁₋₈ alkyl.

In a still further aspect R² is n-hexyl.

In another aspect, R³ is C₅ alkyl-OH, wherein the said C₅ alkyl group isfurther substituted by an additional 3 —OH groups.

In another aspect, R³ is C₆ alkyl-OH, wherein the said C₆ alkyl group isfurther substituted by an additional 4 —OH groups.

In a further aspect R³ is selected from the following:

In another aspect, R³ is selected from the following:

In a further aspect R³ is selected from the following:

In another aspect, R³ is selected from(2S,3R,4R)-2,3,4,5-tetrahydroxypentyl, (2S,3R,4R5R)-2,3,4,5,6-pentahydroxyhexyl,(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl or(2R,3S,4S)-2,3,4,5-tetrahydroxypentyl.

In a further aspect, R³ is selected from(2S,3R,4R)-2,3,4,5-tetrahydroxypentyl, (2S,3R,4R5R)-2,3,4,5,6-pentahydroxyhexyl or(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl.

In another aspect R⁵ is hydrogen.

In a further aspect, R¹ is C₁₋₄ alkyl; m is 1 or 2; A is phenyl orpiperidinyl; X is selected from —C(═O)—, —C(═O)—NR⁴— or —O—C(═O)—NR⁵—; nis 2 or 3; R² is hydrogen or C₁₋₈ alkyl; R³ is C₅₋₆ alkyl-OH, whereinthe said C₅₋₆ alkyl group is further substituted by an additional 3 or 4—OH groups; and R⁴ and R⁵ are both hydrogen.

In a further aspect, R¹ is C₁₋₄ alkyl; m is 1; A is phenyl orpiperidinyl; X is —C(═O)—NH—; n is 2; R² is C₁₋₈ alkyl; R³ is selectedfrom the following:

In a further aspect, R¹ is C₁₋₄ alkyl; m is 1; A is phenyl; X is—C(═O)—NH—; n is 2; R² is C₁₋₈ alkyl; and R³ is selected from thefollowing:

In a further aspect of the invention the compounds of the inventionexhibit R stereochemistry at the carbon centre marked with an asterisk(*) in formula (Ia) below:

An example of a compound of the invention is:

-   3,5-diamino-6-chloro-N-(2-(2-methylbenzyl)-3-(((1-(3-((2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methyl)amino)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl(2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(3-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(3-(hexyl(2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(4-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)butanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(2-(2-methylbenzyl)-3-((1-(3-(2,3,4,5,6-pentahydroxyhexylamino)propanoyl)piperidin-4-yl)methylamino)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(3-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   4-((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)phenyl    2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamate;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl(2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl(2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-(3-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)phenethylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

A further example of a compound of the invention is:

-   3,5-diamino-6-chloro-N-(2-(2-methylbenzyl)-3-(((1-(3-(((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methyl)amino)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(3-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(4-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)butanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(2-(2-methylbenzyl)-3-((1-(3-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)propanoyl)piperidin-4-yl)methylamino)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((1-(3-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   4-((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)phenyl    2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamate;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-((4-((2-(hexyl((2R,3S,4S)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-(3-(3-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)phenethylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

A further example of a compound of the invention is:

-   3,5-diamino-6-chloro-N—((R)-2-(2-methylbenzyl)-3-(((1-(3-(((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methyl)amino)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((1-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((1-(4-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)butanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-2-(2-methylbenzyl)-3-((1-(3-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)propanoyl)piperidin-4-yl)methylamino)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   4-(((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)phenyl    2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamate;-   3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((S)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((S)-3-((4-((2-(hexyl((2R,3S,4S)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N—((R)-3-(3-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)phenethylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

A further feature of the invention is any of the embodiments describedabove with the proviso that any of the specific Examples areindividually disclaimed. For example, a further feature is any of theembodiments described above with the proviso that any of the compoundsselected from the above list of examples of compounds of the inventionare individually disclaimed.

In some embodiments, the compound is a compound of formula (I) excludingat least one compound recited in the Examples below. To illustrate, insome such embodiments, the compound is a compound of formula (I)excluding the compound disclosed in Example X, wherein X may be 1, 2, 3,etc. In other embodiments, the compound is a compound of formula (I)excluding the compounds disclosed in Examples Y, wherein Y may be anycombination of 1, 2, 3, etc.

Compounds of formula (I) may be prepared from compounds of formula (II)according to scheme 1, wherein R¹, R², R³, A, X, m and n are as definedin formula (I), and wherein p=m−1.

Alternatively, compounds of formula (I) may be prepared in a mannerdescribed in scheme 2 wherein R¹, R², R³, A, X, m and n are as definedin formula (I), and wherein Y is halogen, for example, chlorine, bromineor iodine and p=m−1.

Alternatively, compounds of formula (I) may be prepared by theintroduction of R³ in a final step by reductive alkylation of suitablepolyhydroxylated alkylaldehydes such as hexoses or pentoses (theR³-aldehyde), as described in scheme 3, and wherein R¹, R², R³, A, X, mand n are as defined in formula (I). The polyhydroxyalkyl chains presentin R³ may be protected by any suitable alcohol protection groups, forexample as described by P. G. M. Wuts, Th. W. Greene, Greene'sProtective Groups in Organic Synthesis, Wiley-Interscience, New York,2006. Furthermore, as the skilled person will be aware, and as describedin the Examples herein, additional amino groups present in the compoundsof formula (II) may be protected by suitable amino protection groups,for example as described in Wuts and Greene (above), such as bydi-tertbutyl dicarbonate (Boc) or fluorenylmethyloxycarbonyl (FMoc)protecting groups.

Compounds of formula (II) may be prepared enantioselectively by methodsas described in scheme 5 (J. C. D. Müller-Hartwieg, L. La Vecchia, H.Meyer, A. K. Beck, D. Seebach, Org. Synth. 85 (2008), 295.) or by themethod shown in schemes 4, and 6, wherein R¹ is as defined in formula(I).

Schemes 4, 5 and 6 describe access to the (R)-enantiomer of compounds offormula (II). The reactions described in these schemes may equally beapplied to access the related (S)-enantiomer by using starting materialsof reversed absolute configuration or enzymes of different stereospecificity.

Alternatively, compounds of formula (II) with (S)-configuration may beprepared as shown in scheme 7.

The method described at step (a) of Scheme 6 comprises the enzymemediated desymmetrization of diamine (III) using diallylcarbonate.

Suitable enzymes for this reaction include lipases, in particularlipases which originate from Candida Antarctica or Pseudomonas Cepacia.Suitable enzymes include IMM CALB (Candida Antartica Lipase B), IMMCALBY (Candida Antartica Lipase B), Novozym 435 (Candida AntarcticaLipase B), Amano Lipase PS-C1 (Pseudomonas Cepacia Lipase), Amano LipasePS-IM (Pseudomonas Cepacia Lipase) and Amano Lipase PS-D (PseudomonasCepacia Lipase), such as Amano Lipase PS-C1, Amano Lipase PS-D and AmanoLipase PS-IM. The reaction can be carried out in a range of polar andnon-polar solvents, and mixtures thereof, including tert-butyl dimethylether (TBME), tetrahydrofuran (THF), methyl-THF (MeTHF, for example2-methyl-THF), heptanes, cyclohexane and toluene, such as MeTHF. Thereaction can be carried out at a range of temperatures, for example 0°C. to 50° C., for example 15° C. to 35° C., for example about 30° C. Theenantiomeric excess of the resulting crude product can optionally beincreased by resolution processes known to those skilled in the art, forexample by crystallisation from a tartrate salt.

In a further aspect of the invention there is provided a process forpreparing a compound of formula (IV), wherein R¹ is as defined above(such as the compound(R)-allyl-(3-amino-2-(2-methylbenzyl)propyl)carbamate):

comprising the enzyme mediated desymmetrisation of diamine (III) in thepresence of diallycarbonate, and optionally thereafter carrying out anenantiomeric resolution. In a further aspect, the enzyme is selectedfrom one which originates from either Candida Antarctica or PseudomonasCepacia, such as IMM CALB (Candida Antartica Lipase B), IMM CALBY(Candida Antartica Lipase B), Novozym 435 (Candida Antarctica Lipase B),Amano Lipase PS-C1 (Pseudomonas Cepacia Lipase), Amano Lipase PS-IM(Pseudomonas Cepacia Lipase) and Amano Lipase PS-D (Pseudomonas CepaciaLipase). In another aspect, the enzyme is selected from Amano LipasePS-C1, Amano Lipase PS-D and Amano Lipase PS-IM, such as Amino LipasePS-IM.

In another aspect of the invention there is provided the compound(R)-allyl-(3-amino-2-(2-methylbenzyl)propyl)carbamate.

In a further aspect there is provided the use of the compound(R)-allyl-(3-amino-2-(2-methylbenzyl)propyl)carbamate as apharmaceutical intermediate. In a further aspect, there is provided theuse of the compound(R)-allyl-(3-amino-2-(2-methylbenzyl)propyl)carbamate as an intermediatein the manufacture of a compound of formula (I).

Detailed processes to the compounds of the invention are furtherdescribed in the Examples below.

Compounds and salts described in this specification generally may beused in methods to treat various disorders in animals, particularlymammals. Mammals include, for example, humans.

The compounds of the invention, and pharmaceutically acceptable saltsthereof, have activity as pharmaceuticals, in particular as modulatorsof the ENaC, and can be used in the treatment of respiratory tractdisease, diseases of the bone and joints, and other auto-immune andallergic disorders.

Disease states that may be treated with a compound of the invention, ora pharmaceutically acceptable salt thereof, include, but are not limitedto, diseases of the respiratory tract, such as: obstructive diseases ofthe airways including: asthma, including bronchial, allergic, intrinsic,extrinsic, exercise-induced, drug-induced (including aspirin andNSAID-induced) and dust-induced asthma, both intermittent and persistentand of all severities, and other causes of airway hyper-responsiveness;chronic obstructive pulmonary disease (COPD); bronchitis, includinginfectious and eosinophilic bronchitis; emphysema; bronchiectasis;cystic fibrosis; sarcoidosis; farmer's lung and related diseases;hypersensitivity pneumonitis; lung fibrosis, including cryptogenicfibrosing alveolitis, idiopathic interstitial pneumonias, idiopathicpulmonary fibrosis, fibrosis complicating anti-neoplastic therapy andchronic infection, including tuberculosis and aspergillosis and otherfungal infections; complications of lung transplantation; vasculitic andthrombotic disorders of the lung vasculature, and pulmonaryhypertension; antitussive activity including treatment of chronic coughassociated with inflammatory and secretory conditions of the airways,and iatrogenic cough; acute and chronic rhinitis including rhinitismedicamentosa, and vasomotor rhinitis; perennial and seasonal allergicrhinitis including rhinitis nervosa (hay fever); nasal polyposis; acuteviral infection including the common cold, and infection due torespiratory syncytial virus, influenza, coronavirus (including SARS) andadenovirus; acute lung injury; or adult respiratory distress syndrome(ARDS).

In a further aspect of the invention, the diseases of the respiratorytract which may be treated with a compound of the invention, or apharmaceutically salt thereof, includes: chronic obstructive pulmonarydisease (COPD); bronchitis, including infectious and eosinophilicbronchitis; bronchiectasis; cystic fibrosis; antitussive activityincluding treatment of chronic cough associated with inflammatory andsecretory conditions of the airways, and iatrogenic cough; asthma,including bronchial, allergic, intrinsic, extrinsic, exercise-induced,drug-induced (including aspirin and NSAID-induced) and dust-inducedasthma, both intermittent and persistent and of all severities, andother causes of airway hyper-responsiveness.

In a still further aspect of the invention, the disease states which maybe treated with a compound of the invention, or a pharmaceutically saltthereof, includes chronic obstructive pulmonary disease (COPD), cysticfibrosis, asthma, chronic bronchitis and bronchiectasis.

In a yet further aspect of the invention, the disease state which may betreated with a compound of the invention, or a pharmaceutically saltthereof, is chronic obstructive pulmonary disease (COPD).

In one aspect of the invention there is provided a compound of formula(I), or a pharmaceutically acceptable salt thereof, for the treatment ofCOPD.

In a further aspect of the invention there is provided a compound offormula (I), or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament for the treatment of COPD.

In a further aspect of the invention there is provided a method oftreating or preventing COPD in a mammal suffering from, or at risk of,said disease, which comprises administering to a mammal in need of suchtreatment a therapeutically effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof.

In a yet further aspect of the invention, the disease state which may betreated with a compound of the invention, or a pharmaceutically saltthereof, is cystic fibrosis (CF).

In one aspect of the invention there is provided a compound of formula(I), or a pharmaceutically acceptable salt thereof, for the treatment ofCF.

In a further aspect of the invention there is provided a compound offormula (I), or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament for the treatment of CF.

In a further aspect of the invention there is provided a method oftreating or preventing CF in a mammal suffering from, or at risk of,said disease, which comprises administering to a mammal in need of suchtreatment a therapeutically effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof.

When a compound or salt described in this specification is administeredto treat a disorder, a “therapeutically effective amount” is an amountsufficient to reduce or completely alleviate symptoms or otherdetrimental effects of the disorder; cure the disorder; reverse,completely stop, or slow the progress of the disorder; or reduce therisk of the disorder getting worse.

In some embodiments in which a combination therapy is used, the amountof the compound or salt described in this specification and the amountof the other pharmaceutically active agent(s) are, when combined,therapeutically effective to treat a targeted disorder in the animalpatient. In this context, the combined amounts are “therapeuticallyeffective amount” if they are, when combined, sufficient to reduce orcompletely alleviate symptoms or other detrimental effects of thedisorder; cure the disorder; reverse, completely stop, or slow theprogress of the disorder; or reduce the risk of the disorder gettingworse. Typically, such amounts may be determined by one skilled in theart by, for example, starting with the dosage range described in thisspecification for the compound or salt and an approved or otherwisepublished dosage range(s) of the other pharmaceutically activecompound(s).

In order to use a compound of the invention, or a pharmaceuticallyacceptable salt thereof, for the therapeutic treatment of a mammal, suchas human, said ingredient is normally formulated in accordance withstandard pharmaceutical practice as a pharmaceutical composition.Therefore in another aspect the present invention provides apharmaceutical composition which comprises a compound of the formula(I), or a pharmaceutically acceptable salt thereof (active ingredient),and a pharmaceutically acceptable adjuvant, diluent or carrier.

In a further aspect the present invention provides a process for thepreparation of said composition which comprises mixing active ingredientwith a pharmaceutically acceptable adjuvant, diluent or carrier.Depending on the mode of administration, the pharmaceutical compositionwill, for example, comprise from 0.05 to 99% w (percent by weight), suchas from 0.05 to 80% w, for example from 0.10 to 70% w, such as from 0.10to 50% w, of active ingredient, all percentages by weight being based ontotal composition.

The pharmaceutical compositions of this invention may be administered instandard manner for the disease condition that it is desired to treat,for example by topical (such as to the lung and/or airways or to theskin), inhalation, oral, rectal or parenteral administration.

For these purposes the compounds of this invention may be formulated bymeans known in the art. A suitable pharmaceutical composition of thisinvention is one suitable for oral administration in unit dosage form,for example a tablet or capsule which contains between 0.1 mg and 1 g ofactive ingredient.

Each patient may receive, for example, a dose of 0.0001 mgkg⁻¹ to 10mgkg⁻¹, for example in the range of 0.005 mgkg⁻¹ to 5 mgkg⁻¹, of theactive ingredient administered, for example, 1 to 4 times per day.

In an embodiment of the invention, there is provided a pharmaceuticalcomposition comprising a compound of formula (I) in association with apharmaceutically acceptable adjuvant, diluent or carrier, which isformulated for inhaled administration (including oral and nasalinhalation).

The compound of formula (I) may be administered using a suitabledelivery device, for example from a dry powder inhaler, a metered doseinhaler, a nebuliser or a nasal delivery device. Such devices are wellknown.

Dry powder inhalers may be used to administer the compound of formula(I), alone or in combination with a pharmaceutically acceptable carrier,in the later case either as a finely divided powder or as an orderedmixture. The dry powder inhaler may be single dose or multi-dose and mayutilise a dry powder or a powder-containing capsule.

Accordingly in one embodiment, the compound of formula (I), or apharmaceutical composition containing a compound of formula (I), isadministered by means of a dry powder inhaler (DPI).

The DPI may be “passive” or breath-actuated, or “active” where thepowder is dispersed by some mechanism other than the patient'sinhalation, for instance, an internal supply of compressed air. Atpresent, three types of passive dry powder inhalers are available:single-dose, multiple unit dose or multidose (reservoir) inhalers. Insingle-dose devices, individual doses are provided, usually in gelatinecapsules, and have to be loaded into the inhaler before use, examples ofwhich include Spinhaler® (Aventis), Rotahaler® (GlaxoSmithKline),Aeroliser™ (Novartis), Inhalator® (Boehringer) and Eclipse (Aventis)devices. Multiple unit dose inhalers contain a number of individuallypackaged doses, either as multiple gelatine capsules or in blisters,examples of which include Diskhaler® (GlaxoSmithKline), Diskus®(GlaxoSmithKline), Nexthaler® (Chiesi) and Aerohaler® (Boehringer)devices. In multidose devices, drug is stored in a bulk powder reservoirfrom which individual doses are metered, examples of which includeGenuair® (AstraZeneca), Turbuhaler® (AstraZeneca), Easyhaler® (Orion),Novolizer® (ASTA Medica), Clickhaler® (Innovata Biomed), Spiromax®(Teva) and Pulvinal® (Chiesi) devices.

An inhalable pharmaceutical composition for use in a DPI can be preparedby mixing finely divided active ingredient (having an aerodynamicdiameter generally equal to or less than 10 μm, such as equal to or lessthan 5 μm, e.g. from 1 to 5 μm) with a carrier substance, for example, amono-, di- or polysaccharide, a sugar alcohol, or another polyol.Suitable carriers are sugars, for example, lactose, glucose, raffinose,melezitose, lactitol, maltitol, trehalose, sucrose, mannitol; andstarch. Suitably the particles of the active ingredient adhere to thecarrier particles to form an ordered (interactive) powder mixture. Thecarrier particles may have a mass median diameter of from 20 to 1000 m,more usually from 50 to 500 μm.

Alternatively, an inhalable pharmaceutical composition may be preparedby processing a finely divided powder (e.g. consisting of finely dividedactive ingredient and finely divided carrier particles) into spheresthat break up during the inhalation procedure.

The powder mixture may then, as required, be dispensed into hardgelatine capsules, each containing the desired dose of the activeingredient. Alternatively the powder mixture may be loaded into thereservoir of a multidose inhaler for example, the Genuair®, or theTurbuhaler®.

In a further embodiment, the compound of formula (I) is administered bymeans of a metered dose inhaler, particularly a pressurised metered doseinhaler (pMDI). The pMDI contains the active as a suitable solution orsuspension in a pressurised container. The active is delivered byactuating a valve on the pMDI device. Actuation may be manual or breathactuated. In manually actuated pMDIs the device is actuated by the useras they inhale, for example by pressing a suitable release mechanism onthe pMDI device. Breath actuated pMDIs are actuated when the patientinhales through the mouthpiece of the pMDI. This can be advantageous asthe actuation of the device is timed with the patients' inhalation andcan result in a more consistent dosing of the active. Examples of pMDIdevices include for example Rapihaler® (AstraZeneca).

An inhalable pharmaceutical composition for use in a pMDI can beprepared by dissolving or dispersing the compound of formula (I) in asuitable propellant and with or without additional excipients such assolvents (for example ethanol), surfactants, lubricants or stabilisingagents. Suitable propellants include hydrocarbon, chlorofluorocarbon andhydrofluoroalkane (e.g. heptafluoroalkane) propellants, or mixtures ofany such propellants. Preferred propellants are P134a and P227, each ofwhich may be used alone or in combination with other propellants and/orsurfactant and/or other excipients.

In a further embodiment, the compound of formula (I) is administered bymeans of a metered dose inhaler in combination with a spacer. Suitablespacers are well known and include Nebuchamber® (AstraZeneca) orVolumatic® (GlaxoSmithKline).

In a further embodiment, the compound of formula (I) is administered bymeans of a nebuliser. Suitable nebulisers are well known and includeeFlow® (PARI GmbH).

An inhalable pharmaceutical composition for use in a nebuliser can beprepared by dispersing or preferably dissolving the compound of formula(I) in a suitable aqueous medium. The composition may also include forexample suitable pH and/or tonicity adjustment, surfactants andpreservatives. For example a suitable composition for inhalation from anebuliser comprises a compound of formula (I) dispersed in an aqueousmedium (mg/g in Mill-Q water) comprising sodium chloride (9 mg/g);citric acid dried (0.0735 mg/g); sodium citrate (0.19 mg/g);benzalkonium chloride (0.1 mg/g), EDTA (ethylenediamine tetraaceticacid, 0.1 mg/g) and Polysorbate 80 (0.3 mg/g).

In a further embodiment, the compound of formula (I) is administerednasally as a spray from a suitable nasal delivery device, for example aspray pump or an MDI. Alternatively, the compound could be administerednasally as a powder using a suitable DPI device e.g. Rhinocort®,Turbuhaler® (AstraZeneca).

An inhalable pharmaceutical composition for use in a spray pump or MDInasal delivery device can be prepared by dispersing or preferablydissolving the compound of formula (I) in a suitable aqueous medium. Thecomposition may also include for example suitable pH and/or tonicityadjustment, surfactants, preservatives, lubricants flavourings orviscosity modifiers. If required additives to enhance absorption fromthe nasal cavity can be included, such as a suitable bioadhesivepolymer. Suitable dry powder compositions for nasal delivery are ashereinbefore described in relation to DPI delivery. However, where it isdesirable to limit the penetration of the compound into the lung andkeep the compound in the nasal cavity, it may be necessary to use thecompound as larger particle sizes, for example with an average particlediameter greater than about 10 m, e.g. from 10 m to 50 m.

Accordingly, the present invention also provides an inhaler device (forexample a dry powder inhaler, in particular a multiple unit dose drypowder inhaler, or a pMDI inhaler) containing an inhalablepharmaceutical composition of the invention.

The invention further relates to a combination therapy wherein acompound of the invention, or a pharmaceutically acceptable saltthereof, and a second active ingredient are administered concurrently,sequentially or in admixture, for the treatment of one or more of theconditions listed above. Such a combination may be used in combinationwith one or more further active ingredients.

In a further aspect of the present invention there is provided apharmaceutical composition (for example, for use as a medicament for thetreatment of one of the diseases or conditions listed herein, such as CFor COPD) comprising a compound of the invention, or a pharmaceuticallyacceptable salt thereof, and at least one additional active ingredientselected from:

-   -   a) a beta-adrenoceptor agonist;    -   b) a muscarinic receptor antagonist;    -   c) a joint muscarinic receptor antagonist and beta-adrenoceptor        agonist;    -   d) a toll-like receptor agonist (such as a TLR7 or TLR9 agonist)    -   e) an adenosine antagonist;    -   f) a glucocorticoid receptor agonist (steroidal or        non-steroidal);    -   g) a p38 antagonist;    -   h) an IKK2 antagonist;    -   i) a PDE4 antagonist;    -   j) a modulator of chemokine receptor function (such as a CCR1,        CCR2B, CCR5, CXCR2 or CXCR3 receptor antagonist);    -   k) a CRTh2 antagonist; or    -   l) an osmolyte, for example an ionic osmolyte, such as        hypertonic saline as defined below.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a beta-adrenoceptor agonist (including betareceptor subtypes 1-4) such as terbutaline (e.g. as the sulphate salt),salmeterol (e.g. as the xinafoate salt), salbutamol (albuterol) (e.g. asthe sulphate salt), procaterol (e.g. as the hydrochloride salt),pirbuterol (e.g. as the acetate salt), orciprenaline (metaproterenol)(e.g. as the sulphate salt), milveterol (e.g. as the hydrochloridesalt), levosalbutamol (levalbuterol) (e.g. as the hydrochloride salt),abediterol, isoprenaline (isoproterenol) (e.g. as the hydrochloridesalt), indacaterol (e.g. as the maleate salt), vilanterol (e.g. as thetrifenatate (triphenylacetic acid) salt), formoterol (e.g. as thefumarate salt, for example the fumarate dihydrate salt), carmoterol,bitolterol (e.g. as the mesylate salt), olodaterol, bedoradrine (e.g. asthe sulphate salt), bambuterol (e.g. as the hydrochloride salt),arformoterol (e.g. as the tartrate salt), PF-610355(2-[3-[2-[[(2R)-2-hydroxy-2-[4-hydroxy-3-(methanesulfonamido)phenyl]ethyl]amino]-2-methyl-propyl]phenyl]-N-[[3-(4-hydroxyphenyl)phenyl]methyl]acetamide),N-(2-diethylaminoethyl)-N-[2-[2-(4-hydroxy-2-oxo-3H-1,3-benzothiazol-7-yl)ethylamino]ethyl]-3-[2-(1-naphthyl)ethoxy]propanamide,N-cyclohexyl-3-[2-(3-fluorophenyl)ethylamino]-N-[2-[2-(4-hydroxy-2-oxo-3H-1,3-benzothiazol-7-yl)ethylamino]ethyl]propanamide,orN-cyclohexyl-N-[2-[2-(5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl)ethylamino]ethyl]-3-[2-[3-(1-methylpyrazol-4-yl)phenyl]ethoxy]propanamide.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a muscarinic receptor antagonist (for example aM1, M2 or M3 antagonist, such as a selective M3 antagonist) such astiotropium (e.g. as the bromide salt), oxitropium (e.g. as the bromidesalt), ipratropium (e.g. as the bromide salt), glycopyrronium bromide(e.g. as a racemic mixture of stereoisomers, or as the R,R-, R,S-, S,R-,or S,S-stereoisomer, or as a mixture comprising two or more of the R,R-,R,S-, S,R-, or S,S-stereoisomers), aclidinium (e.g. as the bromidesalt), GSK573719(3-[2-[3-(5-cyclohexyloxycarbonyl-thiophen-2-yl)-ureido]-3-(4-hydroxy-phenyl)-propionylamino]-1-(3-hydroxy-benzyl)-1-methyl-piperidinium),BEA2180BR (hydroxy-di-thiophen-2-yl-acetic acid8-methyl-8-aza-bicyclo[3.2.1]oct-6-en-3-yl ester) (e.g. as thehydrobromide salt),[(3R)-1-[2-oxo-2-(2-pyridylamino)ethyl]quinuclidin-1-ium-3-yl]1-phenylcycloheptanecarboxylate(e.g. as the bromide salt), or2-[(4-chlorophenyl)methoxy]ethyl-[[2-[(R)-cyclohexyl-hydroxy-phenyl-methyl]oxazol-5-yl]methyl]-dimethyl-ammonium(e.g. as the napadisylate salt).

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with an agent which is a joint muscarinic receptorantagonist (for example a M1, M2 or M3 antagonist, such as a selectiveM3 antagonist) and a beta-adrenoceptor agonist such as PF4348235([1-[9-[[(2R)-2-hydroxy-2-[4-hydroxy-3-(methanesulfonamido)phenyl]ethyl]amino]nonyl]-4-piperidyl]N-[2-(3-chloro-4-hydroxy-phenyl)phenyl]carbamate),3-[2-[2-chloro-3-[[8-(2-ethylthiazole-4-carbonyl)-11-oxa-3,8-diazaspiro[5.5]undecan-3-yl]methyl]phenyl]ethoxy]-N-cyclopentyl-N-[2-[2-(5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl)ethylamino]ethyl]propanamide,N-butyl-N-[2-[2-(5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl)ethylamino]ethyl]-3-[2-[3-[2-[8-(2-isopropylthiazole-4-carbonyl)-11-oxa-3,8-diazaspiro[5.5]undecan-3-yl]ethyl]phenyl]ethoxy]propanamide,or7-[(1R)-2-[2-[2-fluoro-5-[[8-(2-isopropylthiazole-4-carbonyl)-11-oxa-3,8-diazaspiro[5.5]undecan-3-yl]methyl]phenyl]ethylamino]-1-hydroxy-ethyl]-4-hydroxy-3H-1,3-benzothiazol-2-one.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a toll-like receptor agonist (including anagonist of TLR7 or TLR9) such as loxoribine(7-allyl-2-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-purine-6,8-dione),methyl2-[3-[[3-(6-amino-2-butoxy-8-oxo-7H-purin-9-yl)propyl-(3-morpholinopropyl)amino]methyl]phenyl]acetate(e.g. as the hydrobromide, hydrochloride or dimaleate salt),4-(dimethylamino)butyl2-[4-[[2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl]methyl]phenyl]acetate(e.g. as the disaccharin, difumaric acid, di-1-hydroxy-2-naphthoic acidor mono-benzoic acid salt), or 5′-TCG AAC GTT CGA AGA TGA TGA T(disclosed as SEQ ID 171 in WO2004/0158179).

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with an adenosine antagonist such as regadenoson,ATL-313 (methyl4-[3-[6-amino-9-[(2R,3R,4S,5S)-5-(cyclopropylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl]purin-2-yl]prop-2-ynyl]piperidine-1-carboxylate),or apadenoson (methyl 4-[3-[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl]purin-2-yl]prop-2-ynyl]cyclohexanecarboxylate).

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a glucocorticoid receptor agonist (steroidal ornon-steroidal) such as triamcinolone, triamcinolone acetonide,prednisone, mometasone furoate, loteprednol etabonate, fluticasonepropionate, fluticasone furoate, fluocinolone acetonide, dexamethasonecipecilate, desisobutyryl ciclesonide, clobetasol propionate,ciclesonide, butixocort propionate, budesonide, beclomethasonedipropionate, alclometasone dipropionate,(1R,3aS,3bS,10aR,10bS,11S,12aS)-1-{[(cyanomethyl)thio]carbonyl}-7-(4-fluorophenyl)-11-hydroxy-10a,12a-dimethyl-1,2,3,3a,3b,4,5,7,10,10a,10b,11,12,12a-tetradecahydrocyclopenta[5,6]naphtho[1,2-f]indazol-1-yl2-furoate,(1R,2R,3aS,3bS,10aS,10bR,11S,12aS)-10b-Fluoro-1-{[(fluoromethyl)sulfanyl]carbonyl}-7-(6-fluoropyridin-3-yl)-11-hydroxy-2,10a,12a-trimethyl-1,2,3,3a,3b,4, 5,7,10,10a, 10b,11,12,12a-tetradecahydrocyclopenta[5,6]naphtho[1,2-f]indazol-1-ylmethoxyacetate,2,2,2-trifluoro-N-[(1S,2R)-2-[1-(4-fluorophenyl)indazol-5-yl]oxy-2-(3-methoxyphenyl)-1-methyl-ethyl]acetamide,3-[5-[(1R,2S)-1-(4H-1,3-benzodioxin-7-yl)-2-(2,2-difluoropropanoylamino)propoxy]indazol-1-yl]-N-(3-pyridylmethyl)benzamide,3-[5-[(1R,2S)-2-(2,2-difluoropropanoylamino)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)propoxy]indazol-1-yl]-N-[(3R)-1,1-dioxothiolan-3-yl]benzamide,or3-[5-[(1R,2S)-2-(2,2-difluoropropanoylamino)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)propoxy]indazol-1-yl]-N-[(3R)-tetrahydrofuran-3-yl]benzamide.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a p38 antagonist such as PH797804(3-[3-Bromo-4-(2,4-difluoro-benzyloxy)-6-methyl-2-oxo-2H-pyridin-1-yl]-4,N-dimethyl-benzamide),losmapimod, PF03715455(1-[5-tert-butyl-2-(3-chloro-4-hydroxy-phenyl)pyrazol-3-yl]-3-[[2-[[3-[2-(2-hydroxyethylsulfanyl)phenyl]-[1,2,4]triazolo[4,3-a]pyridin-6-yl]sulfanyl]phenyl]methyl]urea),N-cyclopropyl-4-methyl-3-[6-(4-methylpiperazin-1-yl)-4-oxo-quinazolin-3-yl]benzamide,orN-cyclopropyl-3-fluoro-4-methyl-5-[3-[[1-[2-[2-(methylamino)ethoxy]phenyl]cyclopropyl]amino]-2-oxo-pyrazin-1-yl]benzamide.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a phosphodiesterase (PDE) inhibitor such as amethylxanthanine including theophylline and aminophylline or a selectivePDE isoenzyme inhibitor (including a PDE4 inhibitor or an inhibitor ofthe isoform PDE4D) such as tetomilast, roflumilast, oglemilast,ibudilast, GPD-1116 (3-benzyl-5-phenyl-1H-pyrazolo[4,3-c][1,8]naphthyridin-4-one), ronomilast, NVP ABE 171(4-[8-(2,1,3-benzoxadiazol-5-yl)-1,7-naphthyridin-6-yl]benzoic acid),RPL554(2-[(2E)-9,10-dimethoxy-4-oxo-2-(2,4,6-trimethylphenyl)imino-6,7-dihydropyrimido[6,1-a]isoquinolin-3-yl]ethylurea),CHF5480([(Z)-2-(3,5-dichloro-4-pyridyl)-1-(3,4-dimethoxyphenyl)vinyl](2S)-2-(4-isobutylphenyl)propanoate),or GSK256066(6-[3-(dimethylcarbamoyl)phenyl]sulfonyl-4-(3-methoxyanilino)-8-methyl-quinoline-3-carboxamide).

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with an IKK2 antagonist such as5-(2-isopropylisoindolin-5-yl)-3-[1-(2-methoxyethylsulfonyl)-4-piperidyl]-1H-indole-7-carboxylicacid.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a modulator of chemokine receptor function suchas an antagonist of CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9, CCR10 or CCR11 (for the C—C family), for example aCCR1, CCR2B or CCR5 receptor antagonist; CXCR1, CXCR2, CXCR3, CXCR4 orCXCR5 (for the C—X—C family), for example a CXCR2 or CXCR3 receptorantagonist; or CX₃CR1 for the C—X₃—C family. For example, the presentinvention relates to the combination of a compound of the invention withPS-031291 (pyrrolidine-1,2-dicarboxylic acid2-[(4-chloro-benzyl)-methyl-amide]1-[(4-trifluoromethyl-phenyl)-amide]), CCX-354(1-[4-(4-chloro-3-methoxy-phenyl)piperazin-1-yl]-2-[3-(1H-imidazol-2-yl)pyrazolo[3,4-b]pyridin-1-yl]ethanone),vicriviroc, maraviroc, cenicriviroc, navarixin(2-hydroxy-N,N-dimethyl-3-[[2-[[(1R)-1-(5-methyl-2-furyl)propyl]amino]-3,4-dioxo-cyclobuten-1-yl]amino]benzamide),SB656933(1-(2-chloro-3-fluoro-phenyl)-3-(4-chloro-2-hydroxy-3-piperazin-1-ylsulfonyl-phenyl)urea),N-[1-[(3R)-3-(3,5-difluorophenyl)-3-(1-methylsulfonyl-4-piperidyl)propyl]-4-piperidyl]-N-ethyl-2-(4-methylsulfonylphenyl)acetamide,N-[2-[(2S)-3-[[1-[(4-chlorophenyl)methyl]-4-piperidyl]amino]-2-hydroxy-2-methyl-propoxy]-4-hydroxy-phenyl]acetamide,2-[2-chloro-5-[(2S)-3-(5-chlorospiro[3H-benzofuran-2,4′-piperidine]-1′-yl)-2-hydroxy-propoxy]-4-(methylcarbamoyl)phenoxy]-2-methyl-propanoicacid,N-[2-[(2,3-difluorophenyl)methylsulfanyl]-6-[(1R,2S)-2,3-dihydroxy-1-methyl-propoxy]pyrimidin-4-yl]azetidine-1-sulfonamide,orN-[2-[(2,3-difluorophenyl)methylsulfanyl]-6-[[(1R,2R)-2,3-dihydroxy-1-methyl-propyl]amino]pyrimidin-4-yl]azetidine-1-sulfonamide.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a leukotriene biosynthesis inhibitor,5-lipoxygenase (5-LO) inhibitor or 5-lipoxygenase activating protein(FLAP) antagonist such as TA270(4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone),PF-4191834 (2H-pyran-4-carboxamide,tetrahydro-4-[3-[[4-(1-methyl-1H-pyrazol-5-yl)phenyl]thio]phenyl]-),setileuton, CMI977(1-[4-[(2S,5S)-5-[(4-fluorophenoxy)methyl]tetrahydrofuran-2-yl]but-3-ynyl]-1-hydroxy-urea),fiboflapon(3-[3-tert-butylsulfanyl-1-[[4-(6-ethoxy-3-pyridyl)phenyl]methyl]-5-[(5-methyl-2-pyridyl)methoxy]indol-2-yl]-2,2-dimethyl-propanoicacid), GSK2190915 (1H-indole-2-propanoic acid,3-[(1,1-dimethylethyl)thio]-1-[[4-(6-methoxy-3-pyridinyl)phenyl]methyl]-α,α-dimethyl-5-[(2-pyridinyl)methoxy]-),licofelone, quiflapon(3-[3-tert-butylsulfanyl-1-[(4-chlorophenyl)methyl]-5-(2-quinolylmethoxy)indol-2-yl]-2,2-dimethyl-propanoicacid), veliflapon((2R)-2-cyclopentyl-2-[4-(2-quinolylmethoxy)phenyl]acetic acid), ABT080(4,4-bis[4-(2-quinolylmethoxy)phenyl]pentanoic acid), zileuton,zafirlukast, or montelukast.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a CRTh2 antagonist or a DP2 antagonist such asACT129968(2-[2-[(5-acetyl-2-methoxy-phenyl)methylsulfanyl]-5-fluoro-benzimidazol-1-yl]aceticacid), AMG853(2-[4-[4-(tert-butylcarbamoyl)-2-[(2-chloro-4-cyclopropyl-phenyl)sulfonylamino]phenoxy]-5-chloro-2-fluoro-phenyl]aceticacid), AM211(2-[3-[2-[[benzylcarbamoyl(ethyl)amino]methyl]-4-(trifluoromethyl)phenyl]-4-methoxy-phenyl]aceticacid),2-[4-acetamido-3-(4-chlorophenyl)sulfanyl-2-methyl-indol-1-yl]aceticacid,(2S)-2-[4-chloro-2-(2-chloro-4-ethylsulfonyl-phenoxy)phenoxy]propanoicacid,2-[4-chloro-2-[2-fluoro-4-(4-fluorophenyl)sulfonyl-phenyl]phenoxy]aceticacid, or(2S)-2-[2-[3-chloro-4-(2,2-dimethylpyrrolidine-1-carbonyl)phenyl]-4-fluoro-phenoxy]propanoicacid.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with an MK2 (MAPKAP kinase 2) antagonist such asvaresplabid, PF-3644022((10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8H-[1,4]diazepino[5′,6′:4,5]thieno[3,2-f]quinolin-8-one),or4-benzoyl-D-phenylalanyl-D-seryl-D-tryptophyl-D-seryl-2,3,4,5,6-pentafluoro-D-phenylalanyl-3-cyclohexyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-glutaminyl-D-arginyl-D-arginine.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with a myeloperoxidase antagonist such as resveratrol,piceatannol,3-[[(2R)-tetrahydrofuran-2-yl]methyl]-2-thioxo-7H-purin-6-one, or1-(2-isopropoxyethyl)-2-thioxo-5H-pyrrolo[3,2-d]pyrimidin-4-one.

The present invention still further relates to the combination of acompound of the invention, or a pharmaceutically acceptable saltthereof, together with an osmolyte. As used herein, an “osmolyte” is anosmotically-active small molecule and is intended to cover both ionicand non-ionic osmolytes. In a further aspect of the invention there isprovided a combination of a compound of the invention, or apharmaceutically acceptable salt thereof, together with an ionicosmolyte, such as hypertonic saline.

In a still further aspect of the present invention there is provided apharmaceutical composition (for example, for use as a medicament for thetreatment of one of the diseases or conditions listed herein, such as CFor COPD) comprising a compound of the invention, or a pharmaceuticallyacceptable salt thereof, and at least one additional active ingredientselected from:

-   -   a) a beta-adrenoceptor agonist;    -   b) a muscarinic receptor antagonist;    -   c) a joint muscarinic receptor antagonist and beta-adrenoceptor        agonist; or    -   d) a glucocorticoid receptor agonist (steroidal or        non-steroidal); or    -   e) an osmolyte, for example an ionic osmolyte, such as        hypertonic saline. as defined above.

In a still further aspect of the invention, the at least one additionalactive ingredient is a beta-adrenoceptor agonist. In another aspect, theat least one additional active ingredient is a muscarinic receptorantagonist. In a further aspect, the at least one additional activeingredient is a glucocorticoid receptor agonist (steroidal ornon-steroidal). In a still further aspect, the at least one additionalactive ingredient is a joint muscarinic receptor antagonist andbeta-adrenoceptor agonist. In a still further aspect, the at least oneadditional active ingredient is an osmolyte.

The compounds described in this specification are further illustrated inthe following Examples. These Examples are given by way of illustrationonly and are non-limiting.

ABBREVIATIONS

-   -   aq Aqueous    -   (BOC)₂O/Boc Di-tertbutyl dicarbonate    -   CV Column Volume    -   DEA Diethylamine    -   DEAD Diethylazadicarboxylate    -   DIPA Diisopropylamine    -   DIEA/DIPEA Diisopropylethylamine    -   ESI Electrospray Ionization    -   FA Formic Acid    -   FMOC/Fmoc Fluorenylmethyloxycarbonyl    -   HATU        1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxid hexafluorophosphate    -   HBSS Hanks' Balanced Salt Solution    -   HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid    -   HPLC High Performance Liquid Chromatography    -   IPA Isopropylamine    -   LC/MS Liquid Chromatography-Mass Spectroscopy    -   MeTHF Methyl-tetrahydrofuran    -   MTBE tert-Butyl methyl ether    -   NMP N-methylpyrrolidinone    -   Rt Retention time    -   sat. Saturated    -   SFC Supercritical fluid chromatography    -   TBME tert-Butyl methyl ether    -   TEA Triethylamine    -   THF Tetrahydrofuran    -   wt-% weight-%

General Methods

NMR spectra were recorded on a Bruker Avance, Avance II or Avance IIIspectrometer at a proton frequency of 400, 500 or 600 MHz. A JEOL EX-270spectrometer was used if a proton frequency of 270 MHz is reported. Thecentral peaks of chloroform-δ (H 7.26 ppm), acetone (H 2.04 ppm),dichloromethane-d₂ (H 5.32 ppm), CH₃OD (H 3.30 ppm) or DMSO-d₆ (H 2.49ppm) were used as internal references. Slight variations of chemicalshifts may occur, as is well known in the art, as a result in variationsin sample preparation, such as analyte concentration variations andincluding or omitting additives (for example NMR assay standards ortrifluoroacetic acid).

LC/MS experiments were performed using a Waters Acquity UPLC systemcombined with a Waters Xevo Q-ToF Mass Spectrometer in ESI mode. LC wasrun in two set ups: 1) BEH C18 column (1.7 μm 2.1×50 mm) in combinationwith a gradient (2-95% B in 5 min) of aquous 46 mM ammoniumcarbonate/ammonia buffer at pH 10 (A) and MeCN (B) at a flow rate of 1.0mL/min. 2) HSS C18 column (1.8 μm 2.1×50 mm) with a gradient (2-95% B in5 min) of aquous 10 mM FA/1 mM ammonium formate buffer at pH 3 (A) andMeCN (B) at a flow rate of 1.0 ml/min.

Optical purity, indicated as enantiomeric excess (% ee), was determinedby chiral HPLC using an Agilent 1100 series chromatograph. Method A:System equipped with Chiralpak IC 250×4.6 mm; 5 μm. As mobile phaseheptane/iPrOH/ethanolamine (60:40:0.1) with a flow rate of 1 mL/min wasused. The injection volume was 10 μL and compound detection wasperformed by UV at 268 nm. Method B: System quipped with Chiralpak IC150×4.6 mm, 3 μm. As mobil phase CO₂, 120 bar (A) and MeOH (0.5% DEA)(B) was used in a ratio A/B of 80/20 with a flow rate of 4 mL/min. Theinjection volume was 5 μL.

Preparative HPLC was performed with a Waters FractionLynx system withintregrated MS detection and equipped with Prep C18 OBD 5 m 19×150 mmcolumns from X-Bridge or Sunfire. Alternatively Gilson GX-281 withintregrated UV detection was used, equipped with either Kromasil C8 10μm, 20×250 ID or 50×250 ID mm. As eluent gradients of water/MeCN/AcOH(95/5/0.1) or water/0.05% TFA or water/0.1% NH₄HCO₃ (A) and MeCN (B)were applied.

Preparative SCF was performed with a Waters Prep 100 SCF system withintergrated MS detection, equipped with Waters Viridis 2-EP orPhenomenex Luna Hilic, 30×250 mm, 5 μm. As eluent gradients of CO₂ (100g/min, 120 bar, 40° C.) (A) and MeOH/NH₃ (20 mM) or MeOH (5% FA) or MeOH(B) were applied.

Unless stated otherwise, starting materials were commercially availableor previously described in the literature. All solvents and commercialreagents were of laboratory grade and were used as received unlessotherwise stated.

Chemical names as described in the Examples below were generated usingthe ChemDraw (Ultra 11) naming package. The skilled person will be awarethat different naming packages may produce different chemical names forthe same compound. By way of illustration only, the chemical namesgenerated for Examples 1 to 16 below using the ACD/Name 2012 namingpackage are respectively:

-   3,5-diamino-6-chloro-N-[(2R)-2-(2-methylbenzyl)-3-({[1-(3-{[(2S,3R,4R)-2,3,4,5-tetrahydroxypentyl]amino}propanoyl)piperidin-4-yl]methyl}amino)propyl]pyrazine-2-carboxamide;-   1-deoxy-1-[(2-{[4-({[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}methyl)benzoyl]amino}ethyl)(hexyl)amino]-D-xylitol;-   1-deoxy-1-{[2-({[4-({[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}methyl)piperidin-1-yl]carbonyl}amino)ethyl](hexyl)amino}-D-glucitol;-   3,5-diamino-6-chloro-N-[(2R)-3-({[1-(3-{hexyl[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propanoyl)piperidin-4-yl]methyl}amino)-2-(2-methylbenzyl)propyl]pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-[(2R)-3-({[1-(3-{hexyl[(2S,3R,4R)-2,3,4,5-tetrahydroxypentyl]amino}propanoyl)piperidin-4-yl]methyl}amino)-2-(2-methylbenzyl)propyl]pyrazine-2-carboxamide;-   1-deoxy-1-[{4-[4-({[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}methyl)piperidin-1-yl]-4-oxobutyl}(hexyl)amino]-D-glucitol;-   3,5-diamino-6-chloro-N-[(2R)-2-(2-methylbenzyl)-3-({[1-(3-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propanoyl)piperidin-4-yl]methyl}amino)propyl]pyrazine-2-carboxamide;-   3,5-diamino-6-chloro-N-[(2R)-3-({[1-(3-{hexyl[(2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propanoyl)piperidin-4-yl]methyl}amino)-2-(2-methylbenzyl)propyl]pyrazine-2-carboxamide;-   1-deoxy-1-[(2-{[4-({[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}methyl)benzoyl]amino}ethyl)(hexyl)amino]-D-mannitol;-   1-deoxy-1-[(2-{[4-({[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}methyl)benzoyl]amino}ethyl)(hexyl)amino]-D-glucitol;-   1-deoxy-1-{[2-({[4-({[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}methyl)phenoxy]carbonyl}amino)ethyl](hexyl)amino}-D-glucitol;-   1-deoxy-1-[(2-{[4-(2-{[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}ethyl)benzoyl]amino}ethyl)(hexyl)amino]-D-glucitol;-   1-deoxy-1-[(2-{[4-(2-{[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}ethyl)benzoyl]amino}ethyl)(hexyl)amino]-D-xylitol;-   1-deoxy-1-[(2-{[4-({[(2S)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}methyl)benzoyl]amino}ethyl)(hexyl)amino]-D-xylitol;-   5-deoxy-5-[(2-{[4-({[(2S)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}methyl)benzoyl]amino}ethyl)(hexyl)amino]-D-xylitol;-   1-deoxy-1-[(2-{[3-(2-{[(2R)-3-{[(3,5-diamino-6-chloropyrazin-2-yl)carbonyl]amino}-2-(2-methylbenzyl)propyl]amino}ethyl)benzoyl]amino}ethyl)(hexyl)amino]-D-glucitol.

EXAMPLE 13,5-diamino-6-chloro-N—((R)-2-(2-methylbenzyl)-3-(((1-(3-(((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methyl)amino)propyl)pyrazine-2-carboxamide

Step 1 (R)-(9H-fluoren-9-yl)methyl(3-(4-(((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)piperidin-1-yl)-3-oxopropyl)carbamate

A mixture of(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(318 mg, 0.91 mmol) (Intermediate A), (9H-fluoren-9-yl)methyl3-(4-formylpiperidin-1-yl)-3-oxopropylcarbamate (440 mg, 1.08 mmol)(Intermediate F) and acetic acid (80 μL, 1.40 mmol) in dry THF (6 mL)was stirred at ambient temperature for 30 min. Sodiumtriacetoxyborohydride (340 mg, 1.60 mmol) was added and the reactionmixture was stirred at 40° C. for additional 1.5 h. Water was added andthe mixture stirred for 5 min and the solvents were evaporated off. Theresidual material was partitioned between EtOAc and water. To theaqueous phase was added 10% NaHCO₃ (aq.) until reaching pH 8-9. Thephases were separated and the aqueous phase was backextracted withEtOAc. The combined yellow organic phases were dried over MgSO₄,filtered and the solvents were evaporated off. The crude product waspurified by preparative HPLC (Kromasil C8; H₂O/MeCN/AcOH 95/5/0.2).Relevant fractions were combined and concentrated in vacuo. 10% NaHCO₃(aq.) was added and the water phase was extracted with EtOAc. Theorganic phase was dried over MgSO₄, filtered and evaporated. Theresidual sticky oil was dissolved in DCM/Heptane to give afterevaporation the title compound as a solid, 196 mg (29%, purity 87%).

LC/MS: m/z 739 [M+H]⁺

Step 2(R)-3,5-diamino-N-(3-(((1-(3-aminopropanoyl)piperidin-4-yl)methyl)amino)-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide

(R)-(9H-fluoren-9-yl)methyl3-(4-((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)piperidin-1-yl)-3-oxopropylcarbamate(196 mg, 0.27 mmol) (from step 1) was dissolved in DCM (5 mL) andtreated with piperidine (0.5 mL). The solution was stirred at ambienttemperature for 3 h then evaporated to leave a yellow solid.

The crude material was dissolved in THF (5 mL) and DIPEA (280 μL, 1.60mmol) and treated with (BOC)₂O (0.141 mL, 0.61 mmol) in THF (1 mL). Themixture was stirred at ambient temperature. After 3 h LC/MS showed onlya small conversion to mono-Boc protected product, majority was stillunreacted amine. A large excess of DIPEA and BOC₂(O) were added and themixture was stirred for 1 h at ambient temperature. MeCN (2-3 ml) wasadded to increase solubility and the mixture was heated at 50° C. for 2h then ambient temperature over the weekend.

The volatiles were evaporated in vacuo and the residue partitionedbetween EtOAc and water. The water phase was made acidic by addition of0.5 M citric acid. Brine was added to help separation of the phases. Theorganic phase was washed with water and brine.

Solvent was evaporated off and the residue was dissolved in DCM andpurified by flash chromatography (EtOAc/heptane, gradient 30-100%EtOAc). The solvents were evaporated to afford the di-BOC protectedtitle compound (96 mg).

The obtained di-BOC-protected material (96 mg) was dissolved in DCM (2mL) and treated with TFA (0.5 mL), the solution was stirred at ambienttemperature for 2.5 hours. The mixture was evaporated, residue dissolvedin water and purified by preparative HPLC (Kromasil C8; H₂O/MeCN/HCO₂H95/5/0.2 to 60/40/0.2 over 16 min). Freeze drying afforded the titlecompound as a solid (64 mg, 45%)

LC/MS: m/z 517 [M+H]⁺

¹H NMR (500 MHz, MeOD) δ 1.13-1.34 (m, 2H), 1.83-2.06 (m, 3H), 2.29-2.39(m, 1H), 2.35 (s, 3H), 2.59-2.93 (m, 9H), 3.08-3.23 (m, 3H), 3.32-3.37(m, 1H), 3.67-3.74 (m, 1H), 3.87-3.96 (m, 1H), 4.52-4.6 (m, 1H),7.11-7.22 (m, 4H), 8.51 (s, 0.4H, formic acid residue).

Step 33,5-diamino-6-chloro-N—((R)-2-(2-methylbenzyl)-3-(((1-(3-(((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methyl)amino)propyl)pyrazine-2-carboxamide

(R)-3,5-diamino-N-(3-(((1-(3-aminopropanoyl)piperidin-4-yl)methyl)amino)-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(900 mg, 1.74 mmol) was dissolved in methanol (10 mL).(2R,3S,4R)-2,3,4,5-tetrahydroxypentanal (261 mg, 1.74 mmol) was added,followed by five drops of water. Sodium cyanoborohydride (328 mg, 5.22mmol) was then added and pH adjusted to ˜7.0 by the addition of aceticacid. The resulting solution was stirred at room temperature for 18 h.The volatiles were removed and the resulting crude product was purifiedby preparative HPLC (H2O/MeCN/AcOH 95/5/0.2 to 60/40/0.2 over 20 min).The relevant fractions were collected and freeze-dried. The titlecompound was dissolved in 2M HCl (4 ml) and freeze-dried, a procedurerepeated three times, affording the HCl salt of the title compound (144mg, 11%).

¹H NMR (500 MHz, MeOD) δ 1.30 (ddd, 2H), 1.84-2.13 (m, 3H), 2.32-2.45(m, 4H), 2.62-2.77 (m, 2H), 2.81-2.98 (m, 7H), 3.15 (dd, 1H), 3.26 (d,2H), 3.32-3.42 (m, 2H), 3.58-3.83 (m, 6H), 3.95 (t, 1H), 4.06 (dd, 1H),4.55 (s, 1H), 7.05-7.33 (m, 4H).

EXAMPLE 23,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Method A Step 1 tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl(2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate

(S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate(Intermediate E, 1.23 g, 1.73 mmol),(2R,3S,4R)-2,3,4,5-tetrahydroxypentanal (0.521 g, 3.47 mmol) and DIPEA(0.303 mL, 1.73 mmol) were dissolved in MeOH (10 mL) and stirred at roomtemperature for 1 h. Sodium cyanoborohydride (0.327 g, 5.20 mmol) andacetic acid (0.099 mL, 1.73 mmol) were added and stirring continued at50° C. for 3 days. The reaction was cooled to room temperature, quenchedby addition of 8% NaHCO3 (aq), stirred for 15 min and was thenconcentrated in vacuo. The residue was dissolved in EtOAc (100 mL) and8% NaHCO3 (aq) (50 mL), shaken and the phases separated. The aqueousphase was extracted with EtOAc (100 mL). The combined organic phaseswere dried with Na2SO4 (s), filtered and evaporated in vacuo. Thecompound was purified by preparative HPLC on a Kromasil C8 column (10 μm250×50 ID mm) using a gradient of 20-60% acetonitrile in H2O/MeCN/AcOH95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. The compoundswere detected by UV at 268 nm. The compound was collected andfreeze-dried to yield tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate(1.21 g, 83%) as a white solid.

LC/MS: m/z 843.5 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.74-0.85 (m, 3H), 1.12-1.27 (m, 6H),1.27-1.45 (m, 11H), 2.20 (s, 3H), 2.25-2.35 (m, 1H), 2.39-2.66 (m, 6H),2.9-3.06 (m, 1H), 3.06-3.51 (m, 12H), 3.53-3.6 (m, 1H), 3.6-3.72 (m,1H), 4.2-4.68 (m, 5H), 6.97 (bs, 2H), 7.03-7.19 (m, 6H), 7.72 (d, 2H),7.78-7.99 (m, 1H), 8.21-8.36 (m, 1H).

Step 23,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride

Acetyl chloride (5.69 mL, 80.0 mmol) was added dropwise to an icebathcooled flask of MeOH (20 mL). The mixture was stirred for 5 min andtert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate(1.21 g, 1.43 mmol) was added. The reaction was stirred at roomtemperature for 1.5 h and was then evaporated in vacuo. The residue wasdissolved in water and freeze-dried to yield3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride (1.17 g, 100%) as a pale solid.

LC/MS: m/z 743.4 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.8-0.9 (m, 3H), 1.2-1.33 (m, 6H), 1.62-1.74(m, 2H), 2.26 (s, 3H), 2.3-2.41 (m, 1H), 2.59-2.74 (m, 2H), 2.74-2.85(m, 1H), 2.86-2.98 (m, 1H), 3.09-3.51 (m, 11H), 3.56-3.62 (m, 1H),3.62-3.75 (m, 2H), 3.99-4.09 (m, 1H), 4.14-4.29 (m, 2H), 4.58 (s, 4H),7-7.21 (m, 6H), 7.62 (d, 2H), 7.92 (d, 2H), 8.27 (t, 1H), 8.91-9 (m,1H), 9.16 (d, 2H), 9.52 (d, 1H).

Step 33,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride (1.17 g, 1.43 mmol) was dissolved in water (15 ml) andbasified by addition of 10% Na2CO3 (aq) to pH ˜11. The product wasextracted with EtOAc (5×70 mL). The combined organic phases were driedwith Na₂SO₄ (s), filtered and evaporated in vacuo to yield a semisolid/oil. The residue was dissolved in acetonitrile/water andfreeze-dried to yield3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide(1.02 g, 96%) as a pale solid.

LC/MS: m/z 743.5 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.75-0.85 (m, 3H), 1.12-1.26 (m, 6H),1.31-1.43 (m, 2H), 1.94-2.05 (m, 1H), 2.24 (s, 3H), 2.35-2.49 (m, 5H),2.54-2.65 (m, 4H), 3.14-3.23 (m, 1H), 3.25-3.4 (m, 5H), 3.4-3.49 (m,2H), 3.53-3.59 (m, 1H), 3.62-3.78 (m, 3H), 4.26 (d, 1H), 4.38 (d, 1H),4.45 (t, 1H), 4.51 (d, 1H), 6.96 (bs, 2H), 7.03-7.14 (m, 4H), 7.40 (d,2H), 7.74 (d, 2H), 8.24-8.37 (m, 2H).

Method B3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

To a 5 L flange flask was charged a solution of4-formyl-N-(2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)benzamide(Intermediate I, 133 g, 108.5 g active, 0.26 mol) in EtOH (1500 mL),(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(Intermediate A, 108.8 g, 0.31 mol), EtOH (230 mL) and DIPEA (64.4 mL,0.37 mol). The mixture was heated to 40° C. for 1 h. To the clearsolution was charged AcOH (45.4 ml, 0.79 mol). The solution was stirredat 40° C. for 20 min. To this was charged NaCNBH₃ (23.2 g, 0.37 mol)portionwise over 10 min, noting gas evolution. The mixture was stirredat 40° C. overnight. The reaction was cooled to room temperature,quenched carefully with sat. aq. NaHCO₃ (3100 mL) and stirred at roomtemperature for 1 h. The mixture was extracted with 10% MeOH/EtOAc(2×5000 mL) and 10% MeOH/DCM (5000 mL). The organic phases were combinedand concentrated in vacuo to provide 271 g crude material. NMR analysisindicated ca. 80% purity, with NaCNBH₃ signals present. The residue wasdissolved in DCM (12 L) and IPA (770 ml). The mixture was washed with amixture of sat. aq. NaHCO₃ (2700 ml) and water (770 ml). The aqueousphase was extracted with DCM (1200 ml). The organics were combined andconcentrated in vacuo to provide 298 g (191 g active, 97%) as a yellowsolid. HPLC-MS indicated 88.0% purity. ¹H NMR indicated ca. 60% purity,with ca. 27 wt-% IPA and ca. 8 wt-% Intermediate A.

The so obtained crude (290 g, 235 mmol) was diluted in warm MeOH (750mL) and acetonitrile (15 L) was added continuously while evaporating anazeotrop from of the mixture. After approx. 10 L of solvent wasevaporated the remaining mixture containing a gum like precipitate wasstirred for additional 4 d at room temperature.

The liquid phase was decanted from the gum like residue. To the residue2.5 L of acetonitrile was added and after stirring for 1 h and theacetonitrile was decanted. The so obtained crude (approx. 175 g) wassubjected to a final purification by HPLC chromatography [CelluCoat250×100 mm, 10 μm; mobile phase: MeOH/MeCN/TEA=95:5:0.1; injectedamount: 1.0 g/cycle; cycle time: 3.5 min]. Yield of the title compound:148 g (77%).

LC/MS: m/z 743.5 [M+H]⁺

¹H NMR (600 MHz, DMSO; in case of atropisomeric peaks, only the shift ofthe main atropisomer is reported) δ 0.80 (t, 3H), 1.34-1.4 (m, 2H),1.09-1.24 (m, 6H), 1.34-1.4 (m, 2H), 2.00 (s, 1H), 2.00 (s, 1H), 2.24(s, 3H), 2.36-2.42 (m, 1H), 2.43-2.49 (m, 4H), 2.51-2.54 (m, 1H),2.56-2.64 (m, 4H), 3.15-3.22 (m, 1H), 3.23-3.3 (m, 3H), 3.35-3.4 (m,1H), 3.42 (s, 1H), 3.43-3.49 (m, 1H), 3.54-3.59 (m, 1H), 3.62-3.7 (m,2H), 3.74 (d, 1H), 4.27 (s, 1H), 4.39 (s, 1H), 4.46 (t, 1H), 4.52 (s,1H), 6.96 (s, 1H), 7.03-7.08 (m, 2H), 7.40 (d, 2H), 7.74 (d, 2H),8.26-8.31 (m, 1H), 8.31-8.35 (m, 1H).

¹³C NMR (151 MHz, DMSO) δ 13.91, 19.04, 22.08, 26.48, 26.61, 31.27,34.23, 37.42, 38.60, 41.55, 50.84, 53.06, 53.44, 54.55, 57.43, 62.65,69.23, 71.12, 72.61, 112.95, 117.12, 125.54, 125.83, 126.89, 127.72,129.63, 130.03, 132.85, 135.82, 138.74, 144.02, 152.69, 154.14, 165.55,166.01.

Chiral HPLC (method A; heptane/IPA/TEA 75:25:0.1): 98.9% ee, Rt=14.75min (R), 18.63 min (S).

EXAMPLE 33,5-diamino-6-chloro-N—((R)-3-((1-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Step 1 (9H-fluoren-9-yl)methyl2-(((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethylcarbamate

Into a 100-mL round-bottom flask, was placed 9H-fluoren-9-ylmethylN-(2-aminoethyl)carbamate hydrochloride (1.50 g, 4.71 mmol, 1.0 equiv),(2R,4aR,7R,8R,8aS)-2-phenyl-hexahydro-2H-pyrano[3,2-d][1,3]dioxine-6,7,8-triol(1.51 g, 5.63 mmol, 1.20 equiv), methanol (30 mL). After stirring theabove mixture for 30 min, NaBH₃CN (590 mg, 9.39 mmol, 2.00 equiv), AcOH(570 mg, 9.49 mmol, 2.0 equiv) was added. The resulting solution wasstirred overnight at room temperature. Then hexanal (530 mg, 5.29 mmol,1.50 equiv) was added, followed by more NaBH₃CN (450 mg, 7.16 mmol, 2.00equiv). The resulting solution was stirred for 5 hours at roomtemperature. Then it was concentrated under vacuum. The resultingmixture was washed with of water, extracted with ethyl acetate and theorganic layers combined and dried over Na2SO4. The solids were filteredout. This resulted in 1.6 g (73%) of the title compound as light yellowoil.

LC/MS: m/z 619 [M+H]⁺

¹H NMR (300 MHz, CD₃OD): 1.80-1.92 (m, 3H), 1.15-1.40 (m, 6H), 1.41-1.72(m, 2H), 2.88-3.15 (m, 5H), 3.33-3.43 (m, 2H), 3.55-3.61 (m, 1H),3.67-3.83 (m, 1H), 3.85-4.12 (m, 3H), 4.22-4.40 (m, 4H), 5.57 (s, 1H),7.33-7.65 (9H, m), 7.67 (d, 2H), 7.83 (d, 2H).

Step 2(1R,2S)-3-((2-aminoethyl)(hexyl)amino)-1-[(2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol

Into a 50-mL round-bottom flask, was placed 9H-fluoren-9-ylmethylN-(2-[[(2S,3R)-2,3-dihydroxy-3-[(2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](hexyl)amino]ethyl)carbamate(1.6 g, 2.59 mmol, 1.0 equiv), methanol (10 mL), diethylamine (5 mL).The resulting solution was stirred overnight at room temperature. Theresidue was applied onto a silica gel column and eluted withammonia/MeOH (1:100). This resulted in 600 mg (59%) of the titlecompound as yellow solid.

LC/MS: m/z 397 [M+H]⁺

Step 3 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(2-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethylcarbamoyl)piperidin-4-yl)methyl)carbamate

Into a 50-mL round-bottom flask, was placed(S)-(9H-fluoren-9-yl)methyl-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl(piperidin-4-ylmethyl)carbamate(Intermediate B, 400 mg, 0.60 mmol, 1.0 equiv), triethylamine (177 mg,1.75 mmol, 3.0 equiv), triphosgen (173 mg, 0.58 mmol, 1.0 equiv) in DCM(5 mL). The resulting solution was stirred for 0.5 h at roomtemperature. The resulting mixture was washed with 3×10 mL of water. Theresulting mixture was concentrated under vacuum to give the intermediatechloroformamide which was added dropwise (dissolved in DCM) to asolution of triethylamine (177 mg, 1.75 mmol, 3.0 equiv) and(1R,2S)-3-[(2-aminoethyl)(hexyl)amino]-1-[(2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol(347 mg, 0.88 mmol, 1.50 equiv) in DCM (5 mL) were stirred for 2 h atroom temperature. The resulting mixture was concentrated in vacuo. Theresidue was purified by preparative TLC with DCM/MeOH (10:1). Thisresulted in 145 mg (22%) of the title compound as a yellow solid.

LC/MS: m/z 1090 [M+H]⁺

Step 4 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)piperidin-4-yl)methyl)carbamate

Into a 8-mL vial, was placed (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(2-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethylcarbamoyl)piperidin-4-yl)methyl)carbamate(144 mg, 0.13 mmol, 1.0 equiv), ethanol (0.5 mL), 4M HCl (3 mL). Theresulting solution was stirred overnight at room temperature. Theresulting mixture was concentrated under vacuum. This resulted in 120 mg(crude) of the title compound as yellow oil.

LC/MS: m/z 1002 [M+H]⁺

Step 53,5-diamino-6-chloro-N—((R)-3-((1-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Into a 50-mL round-bottom flask, was placed 9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)piperidin-4-yl)methyl)carbamate(120 mg, 0.12 mmol, 1.0 equiv), diethylamine (2 mL) inN,N-dimethylformamide (4 mL). The resulting solution was stirredovernight at room temperature. The resulting mixture was concentratedunder vacuum. The crude product (100 mg) was purified by Prep-HPLC withthe following conditions (2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column,XBridge Prep C18 OBD Column, 5 um, 19*150 mm; mobile phase, WATER WITH0.05% TFA and ACN (23.0% ACN up to 33.0% in 15 min); Detector, MS,UV 254nm. This resulted in 8 mg (7%) of the title compound as a yellow solid.

LC/MS: m/z 780 [M+H]⁺

¹H NMR (300 MHz, CD₃OD): 0.91-0.95 (m, 3H), 1.27-1.38 (m, 8H), 1.81-1.97(m, 5H), 2.36 (s, 4H), 2.62-2.69 (m, 1H), 2.86-2.96 (m, 7H), 3.31-3.55(m, 3H), 3.63-3.68 (m, 6H), 3.70-3.83 (m, 6H), 4.05-4.17 (m, 3H),7.13-7.20 (m, 4H)

EXAMPLE 43,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Step 1 benzyl3-((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propylamino)propanoate

Into a 100-mL round-bottom flask, was placed 4-methylbenzene-1-sulfonicacid benzyl 3-aminopropanoate (2.00 g, 5.69 mmol, 1.0 equiv),(2R,4aR,7R,8R,8aS)-2-phenyl-hexahydro-2H-pyrano[3,2-d][1,3]dioxine-6,7,8-triol(2.29 g, 8.54 mmol, 1.5 equiv), methanol (30 mL). After 30 min, NaBH₃CN(720 mg, 11.46 mmol, 2.00 equiv), AcOH (680 mg, 11.32 mmol, 2.0 equiv)was added. The resulting solution was stirred overnight at roomtemperature. The resulting mixture was concentrated, diluted with ethylacetate and washed with water. The organic layers were dried overanhydrous sodium sulfate. The solids were filtered off. Removal of thesolvents in vacuo resulted in 1.9 g (77%) the title compound ascolorless oil which was used in next step directly.

LC/MS: m/z 967 [M+H]⁺

Step 2 benzyl3-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)propanoate

Into a 100-mL round-bottom flask, was placed benzyl3-[[(2S,3R)-2,3-dihydroxy-3-[(2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]propanoate(1.90 g, crude), hexanal (660 mg, 6.59 mmol, 1.5 equiv), methanol (30mL). After 30 min, NaBH₃CN (550 mg, 8.75 mmol, 2.0 equiv) and AcOH (530mg, 8.83 mmol, 2.0 equiv) were added. The resulting solution was stirredovernight at room temperature. The resulting mixture was concentrated,diluted with ethyl acetate and washed with water, the organic layerswere dried over anhydrous sodium sulfate. The solids were filtered out.The residue was applied onto a silica gel column and eluted with ethylacetate/petroleum ether (1:2). This resulted in 1.3 g (57%) of benzyl3-[[(2S,3R)-2,3-dihydroxy-3-[(2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](hexyl)amino]propanoateas light yellow oil.

LC/MS: m/z 516 [M+H]⁺

Step 33-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)propanoicacid

Into a 100-mL round-bottom flask, was placed benzyl3-[[(2S,3R)-2,3-dihydroxy-3-[(2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](hexyl)amino]propanoate(1.3 g, 2.52 mmol, 1.0 equiv), Pd/C (5 wt %, 700 mg), ethanol (20 mL).To the above mixture, H₂ (g). The resulting solution was stirred underH₂-atmosphere overnight at room temperature. The solids were filteredoff. The filtrate was concentrated under vacuum. This resulted in 0.5 g(47%) of3-[[(2S,3R)-2,3-dihydroxy-3-[(2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](hexyl)amino]propanoicacid as colorless oil.

LC/MS: m/z 426 [M+H]⁺

Step 4 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate

Into a 50-mL round-bottom flask, was placed3-[[(2S,3R)-2,3-dihydroxy-3-[(2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](hexyl)amino]propanoicacid (381.49 mg, 0.90 mmol, 3.00 equiv), (S)-(9H-fluoren-9-yl)methyl3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl(piperidin-4-ylmethyl)carbamate(Intermediate B, 200.0 mg, 0.30 mmol, 1.0 equiv), HATU (170.5 mg, 0.45mmol, 1.5 equiv), DIEA (57.9 mg, 0.45 mmol, 1.5 equiv),N,N-dimethylformamide (5 mL). The resulting solution was stirred for 2 hat room temperature. Water was added to the above mixture and thesuspension was extracted with ethyl acetate. The organic layers combinedand washed with brine. The mixture was dried over anhydrous sodiumsulfate. The solids were filtered off and the filtrate was evaporated invacuo. The residue was purified by preparative TLC (DCM:MeOH=10:1).Relevant fractions were collected and after removal of the volatilesresulted in 200 mg (62%) of the title compound as a yellow solid.

LC/MS: m/z 1076 [M+H]⁺

Step 5 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate

Into a 50-mL round-bottom flask, was placed (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate(step 4, 200 mg, 0.19 mmol, 1.0 equiv) and solution of HCl (4M inethanol, 2 mL). The resulting solution was stirred overnight at roomtemperature. Evaporation of the volatiles resulted in 200 mg of thecrude title compound as a yellow solid.

LC/MS: m/z 987 [M+H]⁺

Step 63,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Into a 25-mL round-bottom flask, was placed (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate(step 5200 mg, 0.20 mmol, 1.00 equiv), methanol (6 mL), diethylamine (3mL). The resulting solution was stirred overnight at room temperature.The resulting mixture was concentrated under vacuum. The crude product(100 mg) was purified by Prep-HPLC with the following conditions(Prep-HPLC-019): Column, XSelect CSH Prep C18 OBD Column, 5 um, 19*150mm; mobile phase, WATER WITH 0.05% TFA and MeCN (22.0% MeCN up to 30.0%in 8 min, up to 100% in 1 min, hold 100% in 1 min, down to 22% in 2min); Detector, UV 254/220 nm. This resulted in 38 mg (21%) of3,5-diamino-6-chloro-N-[(2R)-3-([[1-(3-[hexyl[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]propanoyl)piperidin-4-yl]methyl]amino)-2-[(2-methylphenyl)methyl]propyl]pyrazine-2-carboxamide;trifluoroacetic acid as a yellow solid.

¹H NMR (300 MHz, CD₃OD): δ 0.95-0.97 (m, 3H), 1.22-1.50 (m, 8H),1.70-2.10 (m, 5H), 2.30-2.51 (m, 4H), 2.63-2.81 (m, 2H), 2.88-3.16 (m,7H), 3.16-3.20 (m, 2H), 3.34-3.78 (m, 5H), 3.51-3.87 (m, 7H), 3.95-4.10(m, 1H), 4.10-4.25 (m, 1H), 4.54 (d, 1H), 7.14-7.22 (m, 4H).

LC/MS: m/z 765 [M+H]⁺

EXAMPLE 53,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Step 1 (S)-(9H-fluoren-9-yl)methyl(1-(3-tert-butoxycarbonylaminopropanoyl)piperidin-4-yl)methyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

A flask (25 mL) was charged with (S)-(9H-fluoren-9-yl)methyl3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl(piperidin-4-ylmethyl)carbamate(Intermediate B, 300 mg, 0.45 mmol),3-(tert-butoxycarbonylamino)propanoic acid (100 mg, 0.53 mmol), DIPEA(145 mg, 1.13 mmol), HATU (342 mg, 0.9 mmol) and DMF (10 mL) was added.The reaction was stirred for 6 h at room temperature. The mixture wasdiluted with 25 mL of ethyl acetate and washed by water (3×25 mL). Theorganic layer was dried over anhydrous Na₂SO₄. The precipitation wasfiltered off and the filtrate was concentrated under vacuum. Theobtained crude product was purified by a silica gel column withdichloromethane/methanol (20:1, v/v) to afford the title compound (280mg, 67%) as light yellow solid.

LC/MS: m/z 839 [M+H]⁺

Step 2 (S)-(9H-fluoren-9-yl)methyl(1-(3-aminopropanoyl)piperidin-4-yl)methyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

Into a 50-mL round-bottom flask was placed (S)-(9H-fluoren-9-yl)methyl(1-(3-tert-butoxycarbonylaminopropanoyl)piperidin-4-yl)methyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(step 1, 216 mg, 0.26 mmol, 1.0 equiv) and hydrogen chloride (4M inmethanol, 5 mL). The resulting solution was stirred for 1 h at roomtemperature. Evaporation of the volatiles resulted in 190 mg of thecrude title compound as a yellow solid.

LC/MS: m/z 739 [M+H]⁺

Step 3 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate

Into a 50-mL round-bottom flask, was placed (S)-(9H-fluoren-9-yl)methyl(1-(3-aminopropanoyl)piperidin-4-yl)methyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(step 2, 190 mg, 0.26 mmol, 1.0 equiv), (3R,4S,5R)-oxane-2,3,4,5-tetrol(39 mg, 0.26 mmol, 1.0 equiv), methanol (6 mL). After 30 min, NaBH₃CN(66 mg, 1.05 mmol, 4.0 equiv) was added slowly at 0° C. The resultingsolution was stirred overnight at room temperature. Then hexanal (39 mg,0.39 mmol, 1.0 equiv) was added, followed by more NaBH3CN (66 mg, 1.05mmol, 4.0 equiv). The resulting solution was stirred for 5 h at roomtemperature. The solvent was removed to give the crude product, whichwas used in the next step directly without further purification.

LC/MS: m/z 957 [M+H]⁺

Step 43,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Into a 50-mL round-bottom flask was placed (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate(step 3, 230 mg, 0.24 mmol, 1.00 equiv), methanol (5 mL), diethylamine(0.5 mL). The resulting solution was stirred for 3 h at roomtemperature. The crude product was purified by Prep-HPLC with thefollowing conditions (Prep-HPLC-019): Column, XSelect CSH Prep C18 OBDColumn, 5 um, 19*150 mm; mobile phase, WATER WITH 0.05% TFA and MeCN(18.0% MeCN up to 35.0% in 8 min, up to 100.0% in 1 min, hold 100.0% in1 min, down to 18.0% in 2 min); Detector, UV 254/220 nm. This resultedin 36.9 mg (16%) of the title compound as a yellow solid.

LC/MS: m/z 735 [M+H]⁺

¹H NMR (300 MHz, CD₃OD): δ 0.96 (d, 3H), 1.26-1.40 (m, 8H), 1.81-2.01(m, 5H), 2.37 (s, 4H), 2.62-2.94 (m, 9H), 3.17-3.47 (m, 7H), 3.55-3.78(m, 6H), 3.98 (d, 1H), 4.15 (s, 1H), 4.56 (d, 1H), 7.14-7.21 (m, 4H).

EXAMPLE 63,5-diamino-6-chloro-N—((R)-3-((1-(4-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)butanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Step 1 (S)-(9H-fluoren-9-yl)methyl((1-(4-((tert-butoxycarbonyl)amino)butanoyl)piperidin-4-yl)methyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

In a 50 mL round-bottomed flask was placed (S)-(9H-fluoren-9-yl)methyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(piperidin-4-ylmethyl)carbamate(Intermediate B, 900 mg, 1.35 mmol),4-((tert-butoxycarbonyl)amino)butanoic acid (821 mg, 4.04 mmol), HATU(2305 mg, 6.06 mmol), and DIEA (1.06 mL, 6.06 mmol) in DMF (10 mL) togive a yellow solution. The resulting solution was stirred at 25° C. for16 h. The reaction mixture was diluted with EtOAc (50 mL), and washedsequentially with water (50 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated in vacuo to afford crude product. Thecrude product was purified by flash chromatography on silica gel, elutedby a gradient of 20 to 70% EtOAc in petroleum ether. Pure fractions wereevaporated to dryness to afford the title compound (834 mg, 73%) as ayellow solid.

LC/MS: m/z 853 [M+H]⁺

Step 2 (S)-(9H-fluoren-9-yl)methyl(1-(4-aminobutanoyl)piperidin-4-yl)methyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

(S)-(9H-fluoren-9-yl)methyl((1-(4-((tert-butoxycarbonyl)amino)butanoyl)piperidin-4-yl)methyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(step 1, 834 mg, 0.98 mmol) was dissolved in a methanolic solution ofHCl (4M) and stirred at room temperature for 1 h. The solvent wasevaporated in vacuo to afford the title compound as a yellow solid.

LC/MS: m/z 753 [M+H]⁺

Step 3 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(4-((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propylamino)butanoyl)piperidin-4-yl)methyl)carbamate

Into a 25-mL round-bottom flask was placed (S)-(9H-fluoren-9-yl)methyl(1-(4-aminobutanoyl)piperidin-4-yl)methyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(step 2, 250 mg, 0.33 mmol, 1.0 equiv), acetal protected D-glucose(2R,4aR,7R,8R,8aS)-2-phenyl-hexahydro-2H-pyrano[3,2-d][1,3]dioxine-6,7,8-triol(133 mg, 0.50 mmol, 1.5 equiv) and methanol (5 mL). The mixture wasstirred for 30 min, then NaBH₃CN (41 mg, 0.65 mmol, 2.0 equiv) and AcOH(40 mg, 0.67 mmol, 2.00 equiv) were added. The resulting solution wasstirred overnight at room temperature. The solvent was removed underreduced pressure to afford 210 mg (crude) of (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(4-((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propylamino)butanoyl)piperidin-4-yl)methyl)carbamateas a yellow solid.

LC/MS: m/z 1005 [M+H]⁺

Step 4 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(4-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)butanoyl)piperidin-4-yl)methyl)carbamate

(9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(4-((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propylamino)butanoyl)piperidin-4-yl)methyl)carbamate(210 mg, crude) was dissolved in MeOH (5 mL), hexanal (42 mg, 0.42 mmol,2.00 equiv) was added, followed by NaBH₃CN (26 mg, 0.41 mmol, 2.00equiv) and AcOH (25 mg, 0.42 mmol, 2.00 equiv). The resulting mixturewas stirred for 2 h at room temperature and then concentrated undervacuum. The residue was taken up with water and extracted with 3×20 mLof ethyl acetate. The combined organic layers and concentrated undervacuum and resulted in 152 mg (67%) of the title compound as a crudeyellow oil.

LC/MS: m/z 1089 [M+H]⁺

Step 5 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(4-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)butanoyl)piperidin-4-yl)methyl)carbamate

Into a 25-mL round-bottom flask was placed (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(4-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)butanoyl)piperidin-4-yl)methyl)carbamate(step 4, 152 mg, 0.14 mmol, 1.00 equiv), HCl (4M in ethanol, 5 mL) andethanol (1 mL). The resulting solution was stirred overnight at roomtemperature. Evaporation of the volatiles resulted in 138 mg of thecrude title compound as yellow oil.

LC/MS: m/z 1002 [M+H]⁺

Step 63,5-diamino-6-chloro-N—((R)-3-((1-(4-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)butanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Into a 25-mL round-bottom flask, was placed (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(4-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)butanoyl)piperidin-4-yl)methyl)carbamate(step 4, 138 mg, 0.14 mmol, 1.0 equiv), diethylamine (2 mL),N,N-dimethylformamide (2 mL). The resulting solution was stirred forovernight at room temperature. The resulting mixture was concentratedunder vacuum. The crude product (110 mg) was purified by preparativeHPLC with the following conditions: Column, XSelect CSH Prep C18 OBDColumn, 5 um, 19*150 mm; mobile phase, WATER WITH 0.05% TFA and MeCN(23% MeCN up to 30% in 8 min, up to 100% in 1 min, hold 100% in 1 min,down to 23.0% in 2 min); Detector, uv 254/220 nm. 15.7 mg (13%) of thetitle compound as trifluoroacetate was obtained as a yellow solid.

LC/MS: m/z 779.5 [M+H]⁺

¹H NMR (300 MHz, CD₃OD): 0.92-0.96 (m, 3H), 1.21-1.39 (m, 8H), 1.78-2.03(m, 7H), 2.36 (s, 4H), 2.61-2.69 (m, 4H), 2.87-2.93 (m, 5H), 3.13-3.31(m, 5H), 3.32-3.36 (m, 1H), 3.63-3.77 (s, 6H), 3.78-3.96 (m, 1H),4.15-4.18 (m, 1H), 4.84-4.86 (m, 1H), 7.13-7.20 (m, 4H).

EXAMPLE 73,5-diamino-6-chloro-N—((R)-2-(2-methylbenzyl)-3-((1-(3-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)propanoyl)piperidin-4-yl)methylamino)propyl)pyrazine-2-carboxamide

Step 1 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propylamino)propanoyl)piperidin-4-yl)methyl)carbamate

Into a 25-mL round-bottom flask was placed3,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide(Example 5/step 2, 20 mg, 0.27 mmol, 1.0 equiv,) and the acetalprotected D-glucose(2R,4aR,7R,8R,8aS)-2-phenylhexahydropyrano[3,2-d][1,3]dioxine-6,7,8-triol(218 mg, 0.81 mmol, 3.0 equiv) in methanol (5 mL). The resultingsolution was stirred for 0.5 h at room temperature. NaBH₃CN (34 mg, 0.54mmol, 2.0 equiv), AcOH (30 mg, 0.50 mmol, 2.0 equiv) was added. Theresulting mixture was stirred overnight at room temperature. Quenchingwith water and extractive work up resulted after removal of the solventsin 220 mg of the crude title compound as a yellow solid.

LC/MS: m/z 992 [M+H]⁺

Step 2 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)propanoyl)piperidin-4-yl)methyl)carbamate

Into a 25-mL round-bottom flask, was placed (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propylamino)propanoyl)piperidin-4-yl)methyl)carbamate(step 1, 200 mg, 0.20 mmol, 1.0 equiv), ethanol (1.5 mL) and HCl (4M inethanol, 5 mL). The resulting solution was stirred overnight at roomtemperature and then concentrated under vacuum. 180 mg of the crudetitle compound was obtained as a yellow solid.

LC/MS: m/z 904 [M+H]⁺

Step 33,5-diamino-6-chloro-N—((R)-2-(2-methylbenzyl)-3-((1-(3-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylamino)propanoyl)piperidin-4-yl)methylamino)propyl)pyrazine-2-carboxamide

Into a 25-mL round-bottom flask was placed 9H-fluoren-9-ylmethylN-[(2S)-3-[(3,5-diamino-6-chloro-3,4-dihydropyrazin-2-yl)formamido]-2-[(2-methylphenyl)methyl]propyl]-N-[[1-(3-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]propanoyl)piperidin-4-yl]methyl]carbamate(step 2, 180 mg, 0.20 mmol, 1.0 equiv), a diethylamine (2 mL) andN,N-dimethylformamide (4 mL). The resulting solution was stirred for 4 hat room temperature and after there was concentrated under vacuum. Thecrude product (150 mg) was purified by preparative HPLC using thefollowing conditions: Column, XBridge Prep C18 OBD Column, 5 um, 19*150mm; mobile phase, water with 0.05% TFA and ACN (16.0% ACN up to 26.0% in15 min); Detector, MS,UV 254/220 nm. This resulted in 15.5 mg (10%) ofthe trifluoroacetate of the title compound as a yellow solid.

LC/MS: m/z 681.4 [M+H]⁺

¹H NMR (300 MHz, CD₃OD): 1.12-1.43 (m, 2H), 1.87-2.12 (m, 3H), 2.14 (s,4H), 2.62-2.75 (m, 2H), 2.87-2.93 (m, 7H), 3.01-3.19 (m, 3H), 3.31-3.37(m, 1H), 3.77-3.80 (m, 5H), 3.87-3.95 (m, 2H), 4.07-4.58 (s, 1H),4.80-4.96 (d, 1H), 7.13-7.20 (m, 4H).

EXAMPLE 83,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Step 1 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-(((2R,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate

Sodium cyanoborohydride (57.0 mg, 0.91 mmol) was added portionwise to amixture of hexanal (45.5 mg, 0.45 mmol) and (9H-fluoren-9-yl)methyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)((1-(3-(((2R,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate[prepared by an analogue procedure as described for Example 7/step 1;starting instead from the acetal protected D-mannitol(2R,4aR,7S,8R,8aS)-2-phenylhexahydropyrano[3,2-d][1,3]dioxine-6,7,8-triol](300 mg, 0.30 mmol) in MeOH (20 mL). The resulting solution was stirredat 25° C. for 4 h. The solvent was removed under reduced pressure. Theresidue was purified by preparative TLC (DCM:MeOH=25:1) to afford(9H-fluoren-9-yl)methyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)((1-(3-(((2R,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate(270 mg, 83%) as a yellow gum.

LC/MS: m/z 1076 [M+H]⁺

¹H NMR (300 MHz, CD₃OD): δ 0.89-1.19 (m, 8H), 1.23-1.37 (m, 11H),1.59-1.65 (m, 4H), 2.16-2.21 (m, 6H), 2.62-3.04 (m, 14H), 3.34-3.68 (m,2H), 3.87-3.96 (m, 4H), 4.11-4.27 (m, 4H), 4.70-4.80 (m, 2H), 5.57 (s,1H), 7.07-7.29 (m, 4H), 7.31-7.50 (m, 7H), 7.50-7.53 (m, 4H), 7.78 (d,2H).

Step 2 (9H-fluoren-9-yl)methyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl((1-(3-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate

(9H-fluoren-9-yl)methyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)((1-(3-(((2R,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate(270 mg, 0.25 mmol) was added to HCl (4M, aq., 15 mL) in ethanol (5 mL).After stirring at 25° C. for 2 h the mixture was concentrated underreduced pressure and afforded the title compound (200 mg, 81%) as ayellow oil.

LC/MS: m/z 987 [M+H]⁺

Step 33,5-diamino-6-chloro-N—((R)-3-((1-(3-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Diethylamine (3 mL) was added to (9H-fluoren-9-yl)methyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)((1-(3-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methyl)carbamate(step 2, 200 mg, 0.20 mmol) in DMF (6 mL). The resulting mixture wasstirred at 25° C. for 2 hours. The solvent was removed under reducedpressure. The crude product was purified by preparative HPLC Column: XBridge C18, 19*150 mm, 5 um; Mobile Phase A:Water/0.05% TFA, MobilePhase B: ACN; Flow rate: 20 mL/min; Gradient: 30% B to 70% B in 10 min;254 nm, as eluents. Fractions containing the desired compound wereevaporated to dryness to afford3,5-diamino-6-chloro-N—((R)-3-(((1-(3-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propanoyl)piperidin-4-yl)methyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide(23.5 mg) as a yellow gum.

LC/MS: m/z 765.4 [M+H]

¹H NMR (300 MHz, CD₃OD): δ 0.94 (t, 3H), 1.29-1.40 (m, 8H), 1.80-2.04(m, 5H), 2.35 (s, 3H), 2.35 (m, 1H), 2.62-2.71 (m, 2H), 2.85-2.97 (m,7H), 3.15-3.30 (m, 4H), 3.31-3.69 (m, 4H), 3.71-3.83 (m, 6H), 4.03-4.06(m, 2H), 4.86-4.87 (m, 1H), 7.13-7.20 (m, 1H).

EXAMPLE 93,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride

Step 1 Tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)carbamate

(S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate(Intermediate E, 500 mg, 0.70 mmol),(3S,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol (254mg, 1.41 mmol) and DIPEA (0.123 mL, 0.70 mmol) were dissolved in MeOH(10 mL) and stirred at room temperature for 1 h. Sodium cyanoborohydride(133 mg, 2.11 mmol) and acetic acid (0.040 mL, 0.70 mmol) were added andstirring continued for 18 h and then at 50° C. for 30 h. The reactionwas cooled to room temperature, quenched by addition of 3 M HCl (aq),stirred for 15 min and was then concentrated in vacuo. The residue wasdissolved in EtOAc (50 mL) and 8% NaHCO₃ (aq) (50 mL), shaken and thephases separated. The aqueous phase was extracted with EtOAc (3×50 mL).The combined organic phases were dried with Na2SO4 (s), filtered andevaporated in vacuo. The compound was purified by preparative HPLC on aKromasil C8 column (10 m 250×50 ID mm) using a gradient of 20-60%acetonitrile in H2O/MeCN/AcOH 95/5/0.2 buffer over 20 minutes with aflow of 100 mL/min. The compounds were detected by UV at 268 nm. Thecompound was collected and freeze-dried to yield the title compound (195mg, 32%) as a colorless solid.

LC/MS: m/z 873.6 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.76-0.85 (m, 3H), 1.13-1.27 (m, 6H),1.3-1.44 (m, 11H), 2.21 (s, 3H), 2.24-2.38 (m, 1H), 2.39-2.53 (m, 4H),2.57-2.66 (m, 2H), 2.77 (d, 1H), 2.91-3.06 (m, 1H), 3.06-3.41 (m, 8H),3.43-3.49 (m, 1H), 3.51 (d, 1H), 3.55-3.65 (m, 3H), 4.23-4.55 (m, 5H),6.97 (bs, 2H), 7.04-7.18 (m, 6H), 7.72 (d, 2H), 7.78-7.99 (m, 1H),8.27-8.37 (m, 1H).

Step 23,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride

To an icebath cooled vial of MeOH (5 mL, 124 mmol) was added acetylchloride (1.42 mL, 20 mmol) dropwise. The mixture was stirred for 5 minand tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)carbamate(190 mg, 0.22 mmol) was then added. The reaction was stirred at roomtemperature for 1.5 h and was then evaporated in vacuo. The residue wasdissolved in water and freeze-dried to yield the title compound (184 mg,100%) as a pale solid.

LC/MS: m/z 773.4 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.81-0.89 (m, 3H), 1.2-1.33 (m, 6H),1.62-1.77 (m, 2H), 2.26 (s, 3H), 2.3-2.41 (m, 1H), 2.6-2.73 (m, 2H),2.75-2.84 (m, 1H), 2.86-2.97 (m, 1H), 3.11-3.27 (m, 3H), 3.27-3.45 (m,5H), 3.45-3.52 (m, 2H), 3.52-3.76 (m, 5H), 3.89-3.99 (m, 1H), 4.03-4.48(m, 7H), 6.88-7.23 (m, 6H), 7.63 (d, 2H), 7.93 (d, 2H), 8.26 (t, 1H),8.93-9.02 (m, 1H), 9.13-9.34 (m, 2H), 9.55 (bs, 1H).

EXAMPLE 103,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Step 1 tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)carbamate

(S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate(Intermediate E, 1.25 g, 1.76 mmol),(3R,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol(0.635 g, 3.52 mmol) and DIPEA (0.308 mL, 1.76 mmol) were dissolved inMeOH (10 mL) and stirred at room temperature for 1 h. Sodiumcyanoborohydride (0.332 g, 5.29 mmol) and acetic acid (0.101 mL, 1.76mmol) were added and stirring continued for 40 h at 50° C. The reactionwas cooled to room temperature, quenched by addition of 8% NaHCO3 (aq),stirred for 30 min and concentrated in vacuo. The residue was dissolvedin EtOAc (100 mL) and 8% NaHCO₃ (aq) (100 mL), shaken and the phasesseparated. The aqueous phase was extracted with EtOAc (2×100 mL). Thecombined organic phases were dried with Na₂SO₄ (s), filtered andevaporated in vacuo. The compound was purified by preparative HPLC on aKromasil C8 column (10 μm 250×50 ID mm) using a gradient of 20-60%acetonitrile in H2O/MeCN/AcOH 95/5/0.2 buffer over 20 minutes with aflow of 100 mL/min. The compounds were detected by UV at 268 nm. Thecompound was collected and freeze-dried to yield tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)carbamate(1.26 g, 82%) as a white solid.

LC/MS: m/z 873.6 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.73-0.86 (m, 3H), 1.1-1.27 (m, 6H),1.27-1.46 (m, 11H), 2.20 (s, 3H), 2.24-2.38 (m, 1H), 2.38-2.49 (m, 3H),2.53-2.69 (m, 3H), 2.9-3.06 (m, 1H), 3.06-3.47 (m, 10H), 3.47-3.54 (m,1H), 3.54-3.76 (m, 3H), 4.18-4.65 (m, 6H), 6.97 (bs, 2H), 7.03-7.19 (m,6H), 7.72 (d, 2H), 7.78-8 (m, 1H), 8.24-8.37 (m, 1H).

Step 23,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride

Acetyl chloride (5.69 mL, 80.0 mmol) was added dropwise to an icebathcooled flask of MeOH (20 mL). The mixture was stirred for 5 min andtert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)carbamate(1.26 g, 1.44 mmol) was then added. The reaction was stirred at roomtemperature for 1.5 h and was then evaporated in vacuo. The residue wasdissolved in water and freeze-dried to yield3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride (1.29 g, 106%) as a pale solid.

LC/MS: m/z 773.4 [M+H]⁺

¹H NMR (500 MHz, DMSO-d₆) δ 0.78-0.89 (m, 3H), 1.18-1.32 (m, 6H),1.62-1.76 (m, 2H), 2.26 (s, 3H), 2.33-2.44 (m, 1H), 2.67 (d, 2H),2.73-2.84 (m, 1H), 2.84-2.96 (m, 1H), 3.1-3.44 (m, 9H), 3.44-3.54 (m,2H), 3.55-3.62 (m, 1H), 3.62-3.75 (m, 3H), 4.02-4.12 (m, 1H), 4.12-4.3(m, 2H), 5.58 (bs, 5H), 6.91-7.21 (m, 6H), 7.64 (d, 2H), 7.94 (d, 2H),8.25 (t, 1H), 8.97-9.06 (m, 1H), 9.28 (bs, 1H), 9.41 (bs, 1H), 9.70 (d,1H).

Step 33,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride (1.23 g, 1.45 mmol) was dissolved in water (15 ml) andbasified by addition of 10% Na2CO3 (aq) to pH ˜11. The product wasextracted with EtOAc (6×70 mL). The combined organic phases were driedwith Na₂SO₄ (s), filtered and evaporated in vacuo to yield a semisolid/oil. The residue was dissolved in acetonitrile/water andfreeze-dried to yield3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide(1.08 g, 96%) as a pale solid.

LC/MS: m/z 773.4 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.75-0.84 (m, 3H), 1.13-1.26 (m, 6H),1.32-1.42 (m, 2H), 1.94-2.05 (m, 1H), 2.24 (s, 3H), 2.34-2.49 (m, 6H),2.54-2.66 (m, 4H), 3.14-3.23 (m, 1H), 3.25-3.35 (m, 4H), 3.35-3.42 (m,1H), 3.42-3.47 (m, 1H), 3.47-3.54 (m, 1H), 3.55-3.61 (m, 1H), 3.61-3.77(m, 4H), 4.26-4.33 (m, 2H), 4.47 (d, 1H), 4.51 (d, 2H), 6.96 (bs, 2H),7.04-7.14 (m, 4H), 7.40 (d, 2H), 7.74 (d, 2H), 8.26-8.37 (m, 2H).

EXAMPLE 114-(((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)phenyl2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamate

Step 1(R)-4-((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)phenylacetate

In a 50 mL pear flask was(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(Intermediate A, 1.6 g, 4.59 mmol) and 4-formylphenyl acetate (0.753 g,4.59 mmol) added to DCM (30 mL) to give a yellow solution. Afterstirring for 1 h sodium triacetoxyborohydride (3.89 g, 18.4 mmol) wasadded and stirring was continued overnight. The mixture was diluted withDCM and washed with water and brine. The organic layer was dried overNa₂SO₄, filtered and evaporated in vacuo to afford crude(R)-4-(((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)phenylacetate (1.70 g, 75%) as a yellow solid. The product was used in thenext step directly without further purification.

LC/MS: m/z 467 [M+H]⁺

Step 2(S)-4-((tert-butoxycarbonyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)phenylacetate

(BOC)₂O (1.07 mL, 4.59 mmol) was added to a mixture of(R)-4-(((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)phenylacetate (1.9 g, 3.82 mmol) and Na₂CO₃ (0.81 g, 7.65 mmol) in the mixturesolvents dioxane (25 mL)/water (8 mL) at 0° C. The mixture was allowedto reach room temperature and stirred overnight. The reaction mixturewas diluted with EtOAc, and washed with saturated brine. The organiclayer was dried over Na₂SO₄, filtered and evaporated in vacu. Theresidue was purified by flash chromatography on silica gel using agradient of 10 to 40% EtOAc in petroleum ether. Pure fractions wereevaporated to dryness to afford the title compound (1.30 g, 57%) as ayellow solid.

LC/MS: m/z 597 [M+H]⁺

¹H NMR (300 MHz, CD₃OD): δ 1.47 (s, 9H), 2.25-2.2.51 (m, 7H), 2.54-2.65(m, 2H), 3.05-3.22 (m, 2H), 3.31-3.39 (m, 2H), 4.13-4.45 (m, 2H),6.93-7.00 (m, 2H), 7.06-7.10 (m, 6H).

Step 3 (S)-tert-butyl3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl(4-hydroxybenzyl)carbamate

NaOH (0.174 g, 4.35 mmol) in water (8 mL) was added dropwise to a cooledsolution of(S)-4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)phenylacetate (1.3 g, 2.18 mmol) in MeOH (20 mL). The reaction solution wasstirred for 3 hours at room temperature. The reaction mixture wasacidified with 2M aq. HCl. The reaction mixture was extracted with EtOAcand the organic phase was washed with water and brine, dried overNa₂SO₄, filtered and evaporated to afford the crude title compound (1.20g, 99%) as yellow solid.

LC/MS: m/z 555 [M+H]⁺

Step 4(S)-4-((tert-butoxycarbonyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)phenylcarbonochloridate

In a 50 mL round-bottomed flask was added (S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-hydroxybenzyl)carbamate(250 mg, 0.45 mmol), DIEA (0.354 mL, 2.03 mmol) to THF (10 mL) to give ayellow solution. The mixture was cooled with an ice bath and triphosgene(200 mg, 0.68 mmol) was added. After stirring for 1 h the reactionmixture was poured into water and extracted with EtOAc (3×20 mL). Theorganic layer was dried over Na₂SO₄, filtered and evaporated to affordthe title compound (250 mg, 90%) as yellow oil.

LC/MS: m/z 617 [M+H]⁺

Step 5 tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-(((2-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethyl)carbamoyl)oxy)benzyl)carbamate

In a 50 mL round-bottomed flask was(1R,2S)-3-((2-aminoethyl)(hexyl)amino)-1-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propane-1,2-diol(Example 3/step 2, 96 mg, 0.24 mmol) and DIEA (0.064 mL, 0.36 mmol)dissolved in THF (10 mL) resulting in a colorless solution.(S)-4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)phenylcarbonochloridate (step 4, 150 mg, 0.24 mmol) was added at 0° C. Theresulting solution was stirred at room temperature for 3 h. The reactionmixture was poured into water (20 mL) and extracted with EtOAc (3×20mL). The organic phases were dried over Na₂SO₄, filtered and evaporatedin vacuo. The residue was purified by preparative TLC (DCM:MeOH=10:1),to afford the title compound (200 mg, 84%) as a yellow solid.

LC/MS: m/z 977 [M+H]⁺

Step 64-(((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)phenyl2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamate

In a 25 mL round-bottomed flask was added tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-(((2-(((2S,3R)-2,3-dihydroxy-3-((2R,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethyl)carbamoyl)oxy)benzyl)carbamate(step 5, 200 mg, 0.20 mmol) to EtOH (1 mL) to give a yellow solution.Aq. HCl (4M, 3 mL) was added and the resulting solution was stirred atroom temperature for 12 h. The crude product was purified by preparativeHPLC (Waters XBridge Prep C18 OBD column, 5i silica, 50 mm diameter, 150mm length), using a gradient of decreasing polarity of water (0.05% TFA)and MeCN as eluent. Fractions containing the desired compound wereevaporated to dryness to afford the title compound (28.0 mg, 13%) as ayellow solid.

LC/MS: m/z 789.4 [M+H]⁺

¹H NMR (300 MHz, CD₃OD): δ 0.79-0.99 (m, 3H), 1.25-1.55 (m, 6H),1.69-1.91 (m, 2H), 2.25-2.45 (m, 4H), 2.51-2.84 (m, 1H), 2.81-3.01 (m,3H,), 3.28-3.31 (m, 4H), 3.34-3.82 (m, 12H), 3.86-3.92 (m, 1H),4.09-4.37 (m, 3H), 7.03-7.35 (m, 6H), 7.453 (d, 2H).

EXAMPLE 123,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride

Step 1 EN06927-61 tert-Butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)carbamate

(S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)phenethyl)carbamate(Intermediate D, 500 mg, 0.69 mmol),(3R,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol (249mg, 1.38 mmol) and DIPEA (0.121 mL, 0.69 mmol) were dissolved in MeOH (5mL) and stirred at 50° C. for 40 min before addition of sodiumcyanoborohydride (130 mg, 2.07 mmol) and acetic acid (0.044 mL, 0.76mmol). Stirring was continued at 50° C. for 21 h and the reactionmixture was then allowed to cool to room temperature. The reaction wasquenched by addition of 8% NaHCO₃ (aq). The mixture was diluted in EtOAc(50 mL) and 8% NaHCO₃ (aq) (50 mL), shaken and the phases separated. Theaqueous phase was extracted with EtOAc (4×50 mL). The combined organicphases were dried with Na2SO4 (s), filtered and evaporated in vacuo. Thecompound was purified by preparative HPLC on a Kromasil C8 column (10 μm250×50 ID mm) using a gradient of 20-60% acetonitrile in H2O/MeCN/AcOH95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. The compoundswere detected by UV at 265 nm. The compound was collected andfreeze-dried to yield the title compound (385 mg, 63%) as a pale solid.

LC/MS: m/z 887.6 [M+H]⁺

¹H NMR (500 MHz, DMSO-d₆) δ 0.80 (t, 3H), 1.12-1.26 (m, 6H), 1.29-1.43(m, 11H), 2.16-2.29 (m, 4H), 2.41-2.48 (m, 2H), 2.56-2.67 (m, 2H), 2.71(t, 2H), 2.96 (dd, 1H), 3.02-3.42 (m, 12H), 3.44 (dd, 1H), 3.47-3.54 (m,1H), 3.55-3.74 (m, 3H), 4.18-4.79 (m, 4H), 6.96 (bs, 2H), 7.06-7.19 (m,6H), 7.72 (d, 2H), 7.8-7.97 (m, 1H), 8.24-8.34 (m, 1H).

Step 23,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride

Acetyl chloride (1.42 mL, 20 mmol) was added dropwise to an icebathcooled solution of MeOH (5 mL, 124 mmol). The solution was stirred for 5min and was then added to tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)carbamate(step 1, 385 mg, 0.43 mmol). The reaction was stirred at roomtemperature for 2.5 h and was then concentrated in vacuo. The residuewas dissolved in water and freeze-dried to yield the title compound (369mg, 99%) as a yellow solid.

LC/MS: m/z 787.4 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.78-0.88 (m, 3H), 1.17-1.31 (m, 6H),1.6-1.74 (m, 2H), 2.23-2.39 (m, 4H), 2.64-2.75 (m, 2H), 2.75-2.87 (m,1H), 2.87-2.99 (m, 1H), 3.05 (t, 2H), 3.09-3.44 (m, 11H), 3.44-3.54 (m,2H), 3.55-3.75 (m, 4H), 4-4.1 (m, 1H), 4.69 (bs, 7H), 6.88-7.2 (m, 5H),7.23-7.3 (m, 1H), 7.36 (d, 2H), 7.88 (d, 2H), 8.24 (t, 1H), 8.85-9.16(m, 3H), 9.63 (d, 1H).

EXAMPLE 133,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride

Step 1 Tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)carbamate

(S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)phenethyl)carbamate(Intermediate D, 500 mg, 0.69 mmol),(2R,3S,4R)-2,3,4,5-tetrahydroxypentanal (208 mg, 1.38 mmol) and DIPEA(0.121 mL, 0.69 mmol) were dissolved in MeOH (5 mL) and stirred at 50°C. for 40 min before addition of sodium cyanoborohydride (130 mg, 2.07mmol) and acetic acid (0.044 mL, 0.76 mmol). Stirring was continued at50° C. for 21 h and was then allowed to cool to room temperature. Thereaction was quenched by addition of 8% NaHCO3 (aq). The mixture wasdiluted in EtOAc (50 mL) and 8% NaHCO3 (aq) (50 mL), shaken and thephases separated. The aqueous phase was extracted with EtOAc (4×50 mL).The combined organic phases were dried with Na2SO4 (s), filtered andevaporated in vacuo. The compound was purified by preparative HPLC on aKromasil C8 column (10 m 250×50 ID mm) using a gradient of 20-60%acetonitrile in H2O/MeCN/AcOH 95/5/0.2 buffer over 20 minutes with aflow of 100 mL/min. The compounds were detected by UV at 265 nm. Thecompound was collected and freeze-dried to yield tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)carbamate(374 mg, 63%) as a pale solid.

LC/MS: m/z 857.6 [M+H]⁺

¹H NMR (500 MHz, DMSO-d₆) δ 0.74-0.85 (m, 3H), 1.11-1.28 (m, 6H),1.28-1.44 (m, 11H), 2.14-2.29 (m, 4H), 2.46 (dd, 2H), 2.55-2.65 (m, 2H),2.71 (t, 2H), 2.96 (dd, 1H), 3.02-3.51 (m, 14H), 3.52-3.6 (m, 1H),3.6-3.71 (m, 1H), 4.17-4.77 (m, 3H), 6.96 (bs, 2H), 7.05-7.2 (m, 6H),7.72 (d, 2H), 7.8-7.97 (m, 1H), 8.23-8.34 (m, 1H).

Step 23,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride

Acetyl chloride (1.42 mL, 20.0 mmol) was added dropwise to an icebathcooled solution of MeOH (5 mL, 123.59 mmol). The solution was stirredfor 5 min and was then added to tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)carbamate(374 mg, 0.44 mmol). The reaction was stirred at room temperature for2.5 h and was then concentrated in vacuo. The residue was dissolved inwater and freeze-dried to yield3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamidedihydrochloride (361 mg, 100%) as a yellow solid.

LC/MS: m/z 757.4 [M+H]⁺

¹H NMR (500 MHz, DMSO-d₆) δ 1-1.09 (m, 3H), 1.39-1.53 (m, 6H), 1.84-1.95(m, 2H), 2.50 (s, 3H), 2.53-2.62 (m, 1H), 2.86-2.97 (m, 2H), 2.97-3.07(m, 1H), 3.09-3.2 (m, 1H), 3.22-3.73 (m, 15H), 3.77-3.96 (m, 3H),4.23-4.32 (m, 1H), 4.84 (bs, 6H), 7.15-7.42 (m, 5H), 7.45-7.52 (m, 1H),7.58 (d, 2H), 8.11 (d, 2H), 8.46 (t, 1H), 9.13-9.2 (m, 1H), 9.30 (d,2H), 9.92 (d, 1H).

EXAMPLE 143,5-diamino-6-chloro-N—((S)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamideacetic acid

Step 1 tert-butyl((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate

(R)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate,(Intermediate H) (163 mg, 0.23 mmol),(2R,3S,4R)-2,3,4,5-tetrahydroxypentanal (69.0 mg, 0.46 mmol) and DIPEA(0.040 mL, 0.23 mmol) were dissolved in MeOH (4 mL) and stirred at roomtemperature for 1 h. Sodium cyanoborohydride (14.44 mg, 0.23 mmol) andacetic acid (0.039 mL, 0.69 mmol) were added and the reaction heated to50° C. for 20 h and was then allowed to cool to room temperature. Thereaction was quenched by addition of 8% NaHCO₃ (aq) and stirred for 30min. The solvent was concentrated in vacuo. The residue was dissolved inEtOAc (25 mL) and 8% NaHCO₃ (aq) (25 mL), shaken and the phasesseparated. The aqueous phase was extracted with EtOAc (2×25 mL). Thecombined organic phases were dried with Na₂SO₄ (s), filtered andevaporated in vacuo. The compound was purified by preparative HPLC on aKromasil C8 column (10 μm 250×20 ID mm) using a gradient of 30-70%acetonitrile in H₂O/ACN/AcOH 95/5/0.2 buffer, over 20 minutes with aflow of 19 mL/min. The compounds were detected by UV at 265 nm. Theproduct fractions were collected and freeze-dried to yield tert-butyl((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate(127 mg, 65.5%) as a solid.

¹H NMR (500 MHz, DMSO-d₆) δ 0.75-0.84 (m, 3H), 1.13-1.26 (m, 6H),1.29-1.42 (m, 11H), 2.20 (s, 3H), 2.25-2.35 (m, 1H), 2.41-2.47 (m, 2H),2.56-2.62 (m, 2H), 2.89-3.05 (m, 1H), 3.07-3.15 (m, 1H), 3.2-3.49 (m,13H), 3.52-3.59 (m, 1H), 3.61-3.69 (m, 1H), 4.21-4.35 (m, 2H), 4.35-4.55(m, 3H), 6.96 (bs, 1H), 7.04-7.19 (m, 6H), 7.71 (d, 2H), 7.77-7.98 (m,1H), 8.24-8.32 (m, 1H).

MS ES⁺: m/z 843.4 [M+H]⁺

Step 23,5-diamino-6-chloro-N—((S)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamideacetic acid

Acetyl chloride (0.711 mL, 10 mmol) was added dropwise to an icebathcooled vial of MeOH (2.5 mL, 61.79 mmol) and stirred for 5 min beforeaddition of tert-butyl((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate(127 mg, 0.15 mmol). The reaction was stirred at room temperature for 1h and was then concentrated in vacuo. The residue was dissolved in EtOAc(25 mL) and 5% Na₂CO₃ (aq) (25 mL), shaken and the phases separated. Theaqueous phase was extracted with EtOAc (5×25 mL). The combined organicphases were dried with Na₂SO₄ (s), filtered and evaporated in vacuo. Thecompound was purified by preparative HPLC on a Kromasil C8 column (10 m250×20 ID mm) using a gradient of 5-45% acetonitrile in H₂O/ACN/AcOH95/5/0.2 buffer, over 20 minutes with a flow of 19 mL/min. The compoundswere detected by UV at 268 nm. The product was freeze-dried to yield3,5-diamino-6-chloro-N—((S)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide(100 mg, 82%) as a pale solid.

¹H NMR (500 MHz, DMSO-d₆) δ 0.77-0.83 (m, 3H), 1.13-1.25 (m, 6H),1.32-1.41 (m, 2H), 1.90 (s, 4H), 1.96-2.05 (m, 1H), 2.24 (s, 3H),2.35-2.55 (m, 6H), 2.55-2.63 (m, 4H), 3.14-3.23 (m, 2H), 3.24-3.4 (m,9H), 3.4-3.49 (m, 4H), 3.56 (q, 1H), 3.62-3.71 (m, 2H), 3.74 (d, 1H),6.96 (bs, 2H), 7.04-7.14 (m, 4H), 7.40 (d, 2H), 7.74 (d, 2H), 8.28 (t,1H), 8.33 (t, 1H).

MS ES⁺: m/z 743.5 [M+H]⁺

EXAMPLE 153,5-diamino-6-chloro-N—((S)-3-((4-((2-(hexyl((2R,3S,4S)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamideacetic acid

Step 1 tert-butyl((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2R,3S,4S)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate

(R)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate,(Intermediate G) (163 mg, 0.23 mmol),(2S,3R,4S)-2,3,4,5-tetrahydroxypentanal (69.0 mg, 0.46 mmol) and DIPEA(40.1 μl, 0.23 mmol) were dissolved in MeOH and stirred at roomtemperature for 1 h. Sodium cyanoborohydride (14.44 mg, 0.23 mmol) andacetic acid (39.5 μl, 0.69 mmol) were added and the reaction heated to50° C. for 20 h and was then allowed to cool to room temperature. Thereaction was quenched by addition of 8% NaHCO₃ (aq) and stirred for 30min. The solvent was concentrated in vacuo. The residue was a dissolvedin EtOAc (25 mL) and 8% NaHCO₃ (aq) (25 mL), shaken and the phasesseparated. The aqueous phase was extracted with EtOAc (5×25 mL). Thecombined organic phases were dried with Na₂SO₄ (s), filtered andevaporated in vacuo. The compound was purified by preparative HPLC on aKromasil C8 column (10 m 250×20 ID mm) using a gradient of 30-70%acetonitrile in H₂O/ACN/AcOH 95/5/0.2 buffer, over 20 minutes with aflow of 19 mL/min. The compounds were detected by UV at 265 nm. Theproduct fractions were collected and freeze-dried to yield tert-butyl((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2R,3S,4S)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate(135 mg, 69.7%) as a solid.

¹H NMR (500 MHz, DMSO-d₆) δ 0.79 (d, 3H), 1.16 (d, 6H), 1.36 (s, 11H),2.20 (s, 3H), 2.31 (s, 1H), 2.50 (dt, 5H), 2.58 (s, 3H), 2.92-3.05 (m,1H), 3.06-3.15 (m, 1H), 3.31 (s, 6H), 3.39-3.5 (m, 2H), 3.53-3.59 (m,1H), 3.61-3.69 (m, 1H), 4.22-4.34 (m, 2H), 4.34-4.55 (m, 3H), 6.97 (bs,2H), 7.04-7.18 (m, 6H), 7.71 (d, 2H), 7.78-7.97 (m, 1H), 8.25-8.32 (m,1H).

MS ES⁺: m/z 843.5 [M+H]⁺

Step 23,5-diamino-6-chloro-N—((S)-3-((4-((2-(hexyl((2R,3S,4S)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamideacetic acid

Acetyl chloride (0.711 mL, 10 mmol) was added dropwise to an icebathcooled vial of MeOH (2.5 mL, 61.79 mmol) and stirred for 5 min beforeaddition of tert-butyl((R)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexyl((2R,3S,4S)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)carbamate(135 mg, 0.16 mmol). The reaction was stirred at room temperature for 1h and was then evaporated in vacuo. The residue was dissolved in EtOAc(25 mL) and 5% Na₂CO₃ (aq) (25 mL), shaken and the phases separated. Theaqueous phase was extracted with EtOAc (5×25 mL). The combined organicphases were dried with Na₂SO₄ (s), filtered and evaporated in vacuo. Thecompound was purified by preparative HPLC on a Kromasil C8 column (10 μm250×20 ID mm) using a gradient of 5-45% acetonitrile in H₂O/ACN/AcOH95/5/0.2 buffer, over 20 minutes with a flow of 19 mL/min. The compoundswere detected by UV at 268 nm. The product was freeze-dried to yield3,5-diamino-6-chloro-N—((S)-3-((4-((2-(hexyl((2R,3S,4S)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide(93 mg, 70.2%) as a pale solid.

¹H NMR (500 MHz, DMSO-d₆) δ 0.76-0.83 (m, 3H), 1.14-1.25 (m, 6H),1.31-1.42 (m, 2H), 1.91 (s, 3H), 1.95-2.06 (m, 1H), 2.24 (s, 3H),2.35-2.54 (m, 6H), 2.60 (dd, 4H), 3.14-3.5 (m, 11H), 3.56 (q, 1H), 3.66(dd, 2H), 3.74 (d, 1H), 6.96 (s, 2H), 7.04-7.14 (m, 4H), 7.40 (d, 2H),7.74 (d, 2H), 8.28 (t, 1H), 8.33 (t, 1H).

MS ES⁺: m/z 743.4 [M+H]⁺

EXAMPLE 163,5-diamino-6-chloro-N—((R)-3-(3-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)phenethylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

Step 1 tert-butyl(S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl(3-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)phenethyl)carbamate

In a 50 mL round-bottomed flask was (S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(3-((2-(hexylamino)ethyl)carbamoyl)phenethyl)carbamate (intermediate D, 370 mg, 0.51 mmol) and(3R,4S,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol (138mg, 0.77 mmol) in MeOH (15 mL) to give a yellow solution. After stirringthe above mixture for 30 min NaCNBH₃ (32.1 mg, 0.51 mmol) was added. Theresulting solution was stirred at 50° C. for 16 hours. The solvent wasremoved under reduced pressure to give crude product tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(3-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)carbamate(500 mg, 110%) as yellow solid. The product was used in the next stepdirectly without further purification.

LC/MS: m/z 887 [M+H]⁺

Step 23,5-diamino-6-chloro-N—((R)-3-(3-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethylcarbamoyl)phenethylamino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide

In a 50 mL round-bottomed flask was tert-butyl((S)-3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(3-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)carbamate(500 mg, 0.56 mmol) in HCl/MeOH (20 mL, 0.56 mmol) to give a yellowsolution. The resulting solution was stirred at rt for 2 h. The solventwas removed under reduced pressure. The crude was purified bypreparative HPLC (Waters XBridge Prep C18 OBD column, 5 i silica, 50 mmdiameter, 150 mm length), using decreasingly polar mixtures of water(containing 0.1% NH₄HCO₃) and MeCN as eluents. Fractions containing thedesired compound were evaporated to dryness to afford3,5-diamino-6-chloro-N—((R)-3-((3-((2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)carbamoyl)phenethyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide(87 mg, 20%) as a yellow solid.

LC/MS: m/z 788 [M+H]⁺

¹H NMR (300 MHz, CD₃OD, 25° C.): δ 0.85-0.91 (m, 3H), 1.15-1.35 (m, 6H),1.39-1.59 (m, 2H), 2.12-2.25 (m, 1H), 2.31 (s, 3H), 2.57-2.89 (m, 13H,3.29-3.33 (m, 2H), 3.38-3.64 (m, 3H), 3.66-3.84 (m, 6H), 7.09-7.16 (m,4H), 7.38 (d, 2H), 7.70-7.73 (m, 2H).

Synthesis of Intermediates Intermediate A(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide

Step 1 Dimethyl 2-(2-methylbenzyl)malonate

A reactor was charged with dimethyl malonate (6.00 kg, 45.4 mol) andmethanol (21.3 L). 25% NaOMe/MeOH (10.37 L, 45.2 mol) was added over 10minutes and then 2-methyl benzylchloride (4.26 kg, 30.3 mol) was addedover 1.5 h, maintaining a reaction temperature below 30° C. Sat. NH₄Cl(53.1 L) was added to the reaction, resulting in pH 8.5. The product wasextracted with isopropyl acetate (3×25 L) and the combined organiclayers were washed with 10% brine (10 L) and concentrated under reducedpressure to afford title compound as an oil (8.63 kg, 68 wt %, 83%). Theso obtained title compound contained dialkylated malonate. Purity byHPLC: 86%.

LC/MS: m/z 237 [M+H]⁺

¹H-NMR (DMSO-d₆, 270 MHz) δ 7.17-7.05 (4H, m), 3.60 (6 h, s), 3.82 (1H,t), 3.12 (2H, s), 2.21 (3H, s); ¹³C-NMR (DMSO-d₆, 270 MHz) δ 169.5,136.6, 136.3, 130.8, 129.5, 127.3, 126.4, 52.8, 52.0, 31.8, 19.4.

Step 2 2-(2-Methylbenzyl)malonamide

A reactor was charged with dimethyl-2-(2-methylbenzyl)malonate (step 1,5.9 kg, 68% wt, 17.0 mol) and methanol (14.8 L). 34% Ammonium hydroxide(24 L, 36 equiv.) was added and the reaction was stirred at 25° C. untilconsidered complete by HPLC analysis. The solids were filtered off andthen MTBE (15.3 L) was added. After 30 min the solids were filtered off,washed with MTBE (2×15.3 L) and dried under reduced pressure at 60° C.to give the title compound as a white solid (3.2 kg, 91%).

LC/MS: m/z 206.9 [M+H]⁺

¹H-NMR (DMSO-d₆, 270 MHz) δ 7.32-7.21 (2H, br s), 7.15-7.00 (6H, m),3.33 (1H, t), 2.96 (2H, d), 2.28 (3H, s); ¹³C-NMR (DMSO-d₆, 270 MHz) δ171.0, 137.5, 136.0, 129.9, 128.9, 126.1, 125.6, 53.2, 32.5, 19.1.

Step 3 2-(2-Methylbenzyl)propane-1,3-diamine

A reactor was charged with 2-(2-methylbenzyl)malonamide (1.32 kg, 6.40mol) and THE (5.4 L). The viscous mixture was cooled to 0° C. and IMborane-THF (25.6 L, 25.6 mol) was added during 2 h, maintaining areaction temperature below 2° C. The reaction was warmed to 15° C. andthen stirred at 50° C. over night. When HPLC analysis indicated lessthan 8% of starting material remained, the reaction mixture was quenchedinto 4M KOH (8 L), maintaining a temperature below 30° C. The organiclayer was concentrated under reduced pressure and the aqueous layer wasextracted twice with MTBE (2×10 L). The combined MTBE extracts was addedto the concentrated organic layer and the combined organic layers werethen charged to a solution of 6M HCl (10 L), maintaining a temperaturebelow 30° C. The aqueous phase was washed with MTBE (10 L) and thenbasified with 6M KOH (12 L). The aqueous phase was extracted twice withTHF (10 L and then 5 L) and the combined organic phase was concentratedunder reduced pressure. Toluene (5 L) was added and then azeotropeddistilled off to remove any residual water. The mixture was trituratedwith toluene (5 L) and the solids were filtered off and washed withtoluene (3 L). The filtrate was concentrated under reduced pressureaffording the title compound as the free amine (1.13 kg, 70% yield).Purity by HPLC: 91%.

LC/MS: m/z 179.0 [M+H]⁺

¹H-NMR (CDCl₃, 270 MHz) δ 7.12-7.00 (4H, m), 2.72-2.59 (4H, m), 2.52(2H, d), 2.27 (3H, s), 1.64 (1H, m), 1.48-1.25 (4H, br s); ¹³C-NMR(CDCl₃, 270 MHz) δ 139.0, 136.3, 130.5, 129.9, 126.2, 125.9, 44.7, 43.8,34.3, 19.6.

Step 4 2-(2-Methylbenzyl)propane-1,3-diamine L-tartrate

To crude diamine (from step 3) (1.62 kg, 9.1 mol) was added ethanol (4L). A solution of L-tartaric acid (1.62 kg, 10.8 mol) dissolved inethanol (11 L) was added, keeping the temperature below 40° C. Themixture was stirred at 25° C. for 3 d and then the solids were filteredoff, washed with ethanol (2×2 L) and dried under reduced pressure at 50°C. to give the title compound as its mono-L-tartrate salt (3.21 kg,quantitative yield). Purity by HPLC: 92.2%.

LC/MS: m/z 179.0 [M+H]⁺

¹H-NMR (D₂O, 270 MHz) δ 7.30-7.15 (4H, m), 4.27 (2H, s), 3.20-3.07 (2H,dd), 3.04-2.94 (2H, dd), 2.79 (2H, d), 2.41 (1H, m), 2.27 (3H, s);¹³C-NMR (D₂O, 270 MHz) δ 178.4, 137.2, 135.7, 131.0, 130.0, 127.6,126.7, 73.9, 40.5, 35.9, 32.8, 18.7.

Step 5 (R)-Allyl (3-amino-2-(2-methylbenzyl)propyl)carbamate (1-(R))

A reactor was charged with 2-(2-methylbenzyl)propane-1,3-diamineL-tartrate salt (3.57 kg, 10.9 mol) and water (17 L). 10M NaOH (17 L,170 mol) was added, maintaining a reaction temperature below 25° C. Themixture was stirred for 30 min at 25° C. for 3 d and then 2-MeTHF (14 L)was added. The layers were separated and the aqueous phase was extractedwith 2-MeTHF (14 L). The combined organic phase was dried (sodiumsulfate), filtered and concentrated under reduced pressure affording thefree diamine. A reactor was charged with the free diamine, 2-MeTHF (37.4L) and diallyl carbonate (1.80 kg, 12.6 mol). Immobilized lipase AmanoPS IM (5.61 kg, 3 wt equivalents) was added in portions over 30 min andthe temperature was set at 30° C. After 4 days, ¹H NMR indicated lessthan 5% starting material left and the reaction mixture was clearfiltered through Celite and concentrated under reduced pressure toafford the title compound (2.68 kg, 82% ee, 100% yield). Purity by HPLC:84.7%.

LC/MS: m/z 263.1 [M+H]⁺

¹H-NMR (CDCl₃, 270 MHz) δ 7.15-7.05 (4H, m), 5.99-5.81 (1H, m),5.80-5.60 (1H, br s), 5.35-5.15 (2H, m), 4.54 (2H, d), 3.40-3.14 (2H,m), 2.85-2.52 (4H, m), 2.29 (3H, s), 1.90-1.77 (1H, m), 1.50-1.10 (2H,br s); ¹³C-NMR (CDCl₃, 270 MHz) δ 156.6, 138.1, 136.1, 133.0, 130.5,129.7, 126.3, 125.9, 117.5, 65.4, 44.0, 43.4, 41.6, 34.2, 19.5.

Step 6 (R)-Allyl (3-amino-2-(2-methylbenzyl)propyl)carbamate (1-(R))D-tartrate salt

Crude (R)-allyl (3-amino-2-(2-methylbenzyl)propyl)carbamate (2.68 kg,10.2 mol) was added to EtOH (7.1 L). A solution of D-tartaric acid (1.53kg, 10.2 mol) in EtOH (8 L) was added keeping the temperature below 25°C. After approximately 1 h a precipitate began to form and the mixturewas allowed to stir over night. MeCN (5 L) was added at 25° C. and thesolids were filtered off, washed with MeCN (2×5 L) and dried underreduced pressure at 40° C. to afford 2.94 kg of the title compound ((R)D-tartrate salt, 100% yield) in a stoichiometric ratio base/di-acid=2:1.Purity by HPLC: 91.6%.

LC/MS: m/z 263.1 [M+H]⁺

¹H-NMR (DMSO-d₆, 270 MHz) δ 7.50 (1H, t), 7.22-7.05 (4H, m), 5.98-5.85(1H, m), 5.35-5.12 (2H, m), 4.48 (2H, d), 3.95 (2H, s), 3.13-2.99 (2H,m), 2.90-2.79 (1H, m), 2.75-2.58 (2H, m), 2.26 (3H, s), 2.08 (1H, m);¹³C-NMR (DMSO-d₆, 270 MHz) δ 174.6, 156.6, 137.4, 136.1, 133.7, 130.3,129.7, 126.3, 125.8, 117.0, 71.8, 64.4, 65.5, 37.6, 32.7, 19.1, 18.9.

Chiral HPLC (method A): 88.3% ee; r.t.=11.14 min (S), 14.57 min (R).

Step 7 (R)-allyl (3-amino-2-(2-methylbenzyl)propyl)carbamate

To a 50 L vessel under nitrogen was added water (14.7 L) and MTBE (14.7L), this was followed by the addition of the (R)-Allyl(3-amino-2-(2-methylbenzyl)propyl)carbamate (1-(R)) D-tartrate salt(step 6, 2940 g). 10M NaOH (14.7 L) was added to the mixture and thetemperature was maintained below 30° C. The mixture was stirred for 30mins, the organics were separated and the aqueous re-extracted with MTBE(14.7 L). A portion of the organics were concentrated in vacuo (1 L) togive an extrapolated yield of 1779 g, 95%.

LC/MS: m/z 263.1 [M+H]⁺

¹H NMR (CDCl₃, 270 MHz) δ 7.05-7.15 (4H, m), 5.80-5.99 (1H, m),5.32-5.14 (m, 2H), 4.52 (d, 2H), 3.15-3.38 (m, 2H), 2.55-2.81 (m, 2H),2.54 (d, 2H), 2.29 (s, 3H), 1.76-1.87 (m, 1H).

Step 8 (S)-allyl tert-butyl(2-(2-methylbenzyl)propane-1,3-diyl)dicarbamate

To a 50 L vessel under nitrogen was added (R)-Allyl(3-amino-2-(2-methylbenzyl)propyl) carbamate (1740 g) dissolved in MTBE(29.4 L). This was followed by the addition of triethylamine (922 mL).The mixture was cooled to 0° C. and treated with BOC-anhydride (1600 mL,1518 g) dissolved in MTBE (3.48 L). The reaction was warmed to 20° C.and stirred for 2 h. The reaction was judged complete by ¹H NMR. Thereaction was charged with water (8.7 L) and stirred for 15 min. Theorganics were separated and washed with 5% citric acid (8.7 L) and brine(8.7 L) before drying over magnesium sulphate, filtering andconcentrating in vacuo affording the title compound (2320 g, 94%). ¹HNMR indicated a purity of >90%.

¹H NMR (CDCl₃, 270 MHz) δ 7.02-7.10 (m, 4H), 5.82-5.99 (m, 1H), 5.55 (t,1H), 5.32-5.15 (m, 2H), 5.02 (t, 1H), 4.52 (d, 2H), 3.17-3.30 (m, 2H),2.95-3.15 (m, 2H), 2.51 (d, 2H), 2.28 (s, 3H0, 1.87-1.95 (m, 1H), 1.43(s, 9H).

Step 9 (S)-tert-butyl (3-amino-2-(2-methylbenzyl)propyl)carbamate

To a 50 L vessel under nitrogen was added (S)-allyl tert-butyl(2-(2-methylbenzyl)propane-1,3-diyl)dicarbamate (2330 g) dissolved inDCM (12.9 L) and MeOH (10.3 L), this was followed by the addition ofPd(PPh₃)₄ (148.4 g). The reaction mixture was cooled to 5° C. andcharged with NaBH₄ (729 g) in portions over a period of 2 h. Thereaction was warmed to 20° C. and stirred for 2 h, The reaction mixturewas carefully quenched into water (46.6 L) and then stirred for 20 min.The organics were separated and the aqueous re-extracted with DCM (23.6L). The combined organics were dried over magnesium sulfate, filteredand concentrated in vacuo to give the title compound (1679 g, 100% yield(˜80% pure by ¹H NMR).

¹H NMR (CDCl₃, 270 MHz) δ 7.02-7.15 (m, 4H), 5.22 (br s, 1H), 3.10-3.28(m, 2H), 2.62-2.80 (m, 2H), 2.52 (d, 2H), 2.29 (s, 3H), 1.75-1.82 (m,1H), 1.92 (s, 9H), 1.30 (br s, 2H).

Step 10 (S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

To a 5 L vessel under nitrogen was added3,5-diamino-6-chloropyrazine-2-carboxylic acid (100 g), HBTU (202.8 g),THF (1200 mL) and DIPEA (111 mL). The reaction was allowed to stir for24 h at room temperature. To the reaction was added (S)-tert-butyl(3-amino-2-(2-methylbenzyl)propyl)carbamate (step 9, 148.6 g) dissolvedin THF (1.49 L). It should be noted that the reaction exothermed by 10°C. during the addition. The reaction was allowed to stir for 24 h atroom temperature, LCMS indicated 5% adduct remaining. The mixture washeated to 35° C. for 4 h. LC/MS indicated 2% adduct remaining. Thereaction was stirred for a further 1 h before being cooled to roomtemperature and concentrated in vacuo. The residue was taken up in ethylacetate (1.5 L) and MTBE (1.5 L) and washed with saturated NaHCO₃ (1 L),5% citric acid (700 mL) and brine (700 mL). The organics were dried overmagnesium sulfate, filtered and concentrated in vacuo. The residue wastaken up in MTBE (1 L) and re-concentrated before being taken up in MTBE(500 mL) and diethyl ether (500 mL) and filtered to remove DIPEA salts.The filtrate was concentrated and analysis indicated some DIPEA saltsremaining. The residue was re-triturated with MTBE (750 mL) and diethylether (750 mL) and again filtered. The filtrate was concentrated to givethe title compound (225 g, 94% (accounting for solvent and TMU).

LC/MS: m/z 349.2 [M+H]⁺

¹H NMR (CDCl₃, 270 MHz) δ 7.46 (t, 1H), 7.13 (m, 4H), 5.52 (t, 1H), 5.22(br s, 2H), 3.62-3.75 (m, 1H), 3.00-3.40 (m, 5H), 2.80 (s, 3H),2.49-2.75 (m, 2H), 1.95-2.02 (m, 1H), 1.42 (s, 9H).

Step 113,5-Diamino-N-[(2R)-3-amino-2-(2-methylbenzyl)propyl]-6-chloropyrazine-2-carboxamide

To a 20 L flange flask under nitrogen was added (S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate (step 10, 2215 g) in dioxane (6 L). The mixture was stirredfor 20 min until complete dissolution. To the reaction was then added 4MHCl in dioxane (10 L). The reaction evolved a considerable amount of gasand the temperature rose to 35° C. before cooling was applied. A viscousoily precipitate was observed in the reaction. LC/MS of the reactionmixture after 1 h indicated complete reaction. The supernatant liquorwere decanted off and concentrated in vacuo. The oily viscous solid wasdissolved in water (6 L) and combined with the concentrated supernatantdissolved in water (2 L). The aqueous was washed with MTBE (5 L). Theaqueous was then basified with 10 M NaOH (2 L) and extracted with2-Me-THF (4×10 L). The organics were dried, filtered and concentrated invacuo. The residue was then concentrated from MTBE (2×4 L) to removeresidual 2-MeTHF. The solid was triturated with MTBE:heptane (9 L: 1.8L) and allowed to stir over the weekend. The solid was filtered anddried in the oven at 40° C. affording the title compound (1657 g, 96%).

¹H NMR (CDCl₃, 270 MHz) δ 7.60 (t, 1H), 7.08-7.15 (m, 4H), 5.75 (br s,2H), 5.12 (b s, 2H), 3.38-3.45 (m, 2H), 2.52-2.82 (m, 4H), 2.30 (s, 3H),1.80-1.98 (m, 2H), 1.20-1.28 (m, 2H).

Step 12(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(2R,3S)-2-benzoyl-3-(benzoyloxy)succinate

To a 5 L vessel under nitrogen was added3,5-Diamino-N-[(2R)-3-amino-2-(2-methylbenzyl)propyl]-6-chloropyrazine-2-carboxamide(127.4 g) and methanol (892 mL). The mixture was gently heated to 30° C.until all the solid had dissolved. The reaction was cooled to roomtemperature and (+)-dibenzoyl-D-tartaric acid (65.5 g) in methanol (458mL) was added. The reaction was allowed to stir, after ˜2 h solid beganto precipitate. The reaction was allowed to stir over the weekend.Isopropyl acetate (1.5 L) was added to the mixture and stirring wascontinued for 4 h. The precipitate was then filtered off and washed withisopropyl acetate (2×1 L). The solid was then dried in the oven for 3 dat 40° C. A total of 147.5 g (74%) was obtained in the stoichiometricratio base/di-acid=2:1.

¹H NMR (CDCl₃, 270 MHz): δ 7.60 (t, 1H), 7.08-7.15 (m, 4H), 5.75 (br s,2H), 5.12 (b s, 2H), 3.38-3.45 (m, 2H), 2.52-2.82 (m, 4H), 2.30 (s, 3H),1.80-1.98 (m, 2H), 1.20-1.28 (m, 2H).

Chiral HPLC (method A): 98.7% ee, Rt=11.93 min (R), 14.24 min (S).

Step 13(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide

(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(2R,3S)-2-benzoyl-3-(benzoyloxy)succinate (20 g, 38.46 mmol) wassuspended in 2-methyl-THF (200 mL) and water (200 mL) and cooled with anicebath. Sodium hydroxide, 1 M (42.3 mL, 42.31 mmol) was added slowlyand the mixture was then stirred at 40° C. until all material haddissolved. The mixture was cooled to room temperature and the phaseswere separated. The aqueous phase was extracted with 2-methyl-THF (2×200mL). The combined organic phases were washed with brine (200 mL), driedwith a phase separator and evaporated in vacuo to yield(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(13.3 g, 99%) as a pale solid.

LC/MS: m/z: 349.3 [M+H]⁺.

1H NMR (500 MHz, DMSO-d6) δ 1.50 (bs, 2H), 1.76-1.87 (m, 1H), 2.26 (s,3H), 2.4-2.49 (m, 2H), 2.52-2.62 (m, 2H), 3.18-3.28 (m, 2H), 6.95 (bs,2H), 7.04-7.17 (m, 4H), 8.14 (t, 1H).

Chiral HPLC (method B): 99.3% ee, Rt=11.55 min (S), 13.28 min (R).

[α]_(D) ²⁰: +11.8 (c 1.0, MeCN).

Intermediate B (S)-(9H-fluoren-9-yl)methyl3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl(piperidin-4-ylmethyl)carbamate

Step 1 (R)-tert-butyl4-((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)piperidine-1-carboxylate

Into a 50-mL round-bottom flask, was placed a mixture of tert-butyl4-formylpiperidine-1-carboxylate (122 mg, 0.57 mmol, 1.00 equiv) and3,5-diamino-N-[(2R)-3-amino-2-[(2-methylphenyl)methyl]propyl]-6-chloropyrazine-2-carboxamide(Intermediate A, 200 mg, 0.57 mmol, 1.0 equiv) in dichloromethane (5mL), the mixture was stirred for 1 h. Then sodium triacetoxyborohydride(486 mg, 2.29 mmol, 4.00 equiv) was added. The resulting solution wasstirred overnight at room temperature. The resulting mixture was washedwith 3×10 mL of water, dried over anhydrous sodium sulfate andconcentrated under vacuum. This resulted in 250 mg (80%) of the titlecompound as a yellow solid.

LC/MS: m/z: 546 [M+H]⁺.

Step 2 (S)-tert-butyl4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)piperidine-1-carboxylate

FMOC-chloride (19 mg, 0.07 mmol, 1.00 equiv) in dioxane (2.0 mL) wasadded dropwise to a mixture of (R)-tert-butyl4-((3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propylamino)methyl)piperidine-1-carboxylate(step 1, 40 mg, 0.07 mmol, 1.0 equiv) and Na₂CO₃ (12 mg, 0.11 mmol, 1.5equiv) in water/dioxane (1:2, 3 mL). The resulting solution was stirredfor 2 h at room temperature, extracted with 3×5 mL of ethyl acetate. Thecombined organic phases were dried over anhydrous sodium sulfate. Afterremoval of the solvents in vacuo 42 mg (75%) of the title compound wasobtained as a yellow solid.

LC/MS: m/z: 768 [M+H]⁺

Step 3 (S)-(9H-fluoren-9-yl)methyl3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl(piperidin-4-ylmethyl)carbamate

Into a 25-mL round-bottom flask was placed a solution of (S)-tert-butyl4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)piperidine-1-carboxylate(step 2, 20 mg, 0.03 mmol, 1.0 equiv) in HCl/MeOH (4M, 3 mL). Theresulting solution was stirred for 1 h at room temperature. Theresulting mixture was concentrated under vacuum. This resulted in 15 mg(86%) of 9H-fluoren-9-ylmethylN-[(2S)-3-[(3,5-diamino-6-chloropyrazin-2-yl)formamido]-2-[(2-methylphenyl)methyl]propyl]-N-(piperidin-4-ylmethyl)carbamateas a yellow solid.

LC/MS: m/z: 668 [M+H]⁺

Intermediate C (9H-fluoren-9-yl)methyl (2-aminoethyl)(hexyl)carbamatehydrochloride

Step 1 tert-butyl (2-(benzyl(hexyl)amino)ethyl)carbamate

N-benzylhexan-1-amine (2.69 g, 14.1 mmol), tert-butyl(2-oxoethyl)carbamate (4.48 g, 28.1 mmol) and DIPEA (2.46 mL, 14.1 mmol)was dissolved in MeOH (20 mL) and stirred at room temperature for 45min. Sodium cyanoborohydride (1.77 g, 28.1 mmol) and acetic acid (0.806mL, 14.1 mmol) were added and stirring continued for 18 h. The reactionwas quenched by addition of 8% NaHCO₃ (aq) and stirred at roomtemperature for 1 h. The solvent was concentrated in vacuo. The residuewas diluted with water and extracted with EtOAc (2×100 mL). The combinedorganic phases were dried with Na2SO4 (s), filtered and evaporated invacuo. The residue was purified by automated flash chromatography on twoBiotage® KP-SIL 100 g columns. A gradient from 5-50% of EtOAc in heptaneover 12 CV was used as mobile phase. The product was collected andevaporated in vacuo to yield tert-butyl(2-(benzyl(hexyl)amino)ethyl)carbamate (3.19 g, 68%) as a colorless oil.

LC/MS: m/z: 335.6 [M+H]⁺

1H NMR (500 MHz, CDCl₃) δ 0.84-0.92 (m, 3H), 1.19-1.35 (m, 6H),1.38-1.66 (m, 11H), 2.43 (t, 2H), 2.52 (t, 2H), 3.09-3.21 (m, 2H), 3.56(s, 2H), 4.83-4.93 (m, 1H), 7.22-7.37 (m, 5H).

Step 2 tert-butyl (2-(hexylamino)ethyl)carbamate

tert-butyl (2-(benzyl(hexyl)amino)ethyl)carbamate (3.19 g, 9.54 mmol)was dissolved in MeOH (25 mL) and 20% Pd(OH)₂/C (0.201 g, 0.29 mmol)added. The reaction was hydrogenated in a Buchi hydrogenator at 4 barand room temperature for 18 h. The reaction had stopped. More 20%Pd(OH)₂/C (200 mg, 0.28 mmol) was added and hydrogenation continued for6 h, hydrogenation had stopped. The catalyst was filtered off, washedwith MeOH and the filtrate concentrated in vacuo. The residue wasdissolved in MeOH (15 mL), 20% Pd(OH)₂/C (250 mg, 0.36 mmol) was addedand hydrogenation continued at 4 bar and room temperature for 16 h. Thecatalyst was filtered off, washed with MeOH and the filtrate evaporatedin vacuo. The reaction was once again dissolved in MeOH, 20% Pd(OH)₂/C(250 mg, 0.36) added and hydrogenation continued for 3 days. Thecatalyst was filtered off, washed with MeOH and the filtrate evaporatedin vacuo to yield tert-butyl (2-(hexylamino)ethyl)carbamate (2.38 g,102%) as a colorless oil.

¹H NMR (500 MHz, CDCl₃) δ 0.89 (t, 3H), 1.22-1.36 (m, 6H), 1.41-1.53 (m,11H), 2.61 (t, 2H), 2.75 (t, 2H), 3.19-3.28 (m, 2H), 4.97 (bs, 1H).

Step 3 (9H-fluoren-9-yl)methyl(2-((tert-butoxycarbonyl)amino)ethyl)(hexyl)carbamate

To an icebath cooled solution of tert-butyl(2-(hexylamino)ethyl)carbamate (2.38 g, 9.74 mmol) in DCM (40 mL) wereadded DIPEA (1.79 mL, 10.2 mmol) and (9H-fluoren-9-yl)methylcarbonochloridate (2.65 g, 10.2 mmol). The reaction was stirred withcooling for 10 min and then at room temperature for 1 h. Water (20 mL)was added, shaken and the phases separated. The aqueous phase wasextracted with DCM (2×20 mL). The combined organic phases were driedwith a phase separator and evaporated in vacuo. The residue was purifiedby automated flash chromatography on a Biotage® KP-SIL 100 g column. Agradient from 5-30% of EtOAc in heptane over 9 CV followed by 30% EtOAcin heptane over 3 CV were used as mobile phase. The product wascollected using the wavelength 264 nm. The collected fractions wereevaporated in vacuo to yield (9H-fluoren-9-yl)methyl(2-((tert-butoxycarbonyl)amino)ethyl)(hexyl)carbamate (3.70 g, 81%) as acolorless oil.

LC/MS: m/z: 467.4 [M+H]⁺

¹H NMR (500 MHz, CDCl₃) δ 0.89 (t, 3H), 1.06-1.36 (m, 8H), 1.43 (s, 9H),2.78-2.87 (m, 1H), 2.95-3.08 (m, 2H), 3.11-3.36 (m, 3H), 4.18-4.26 (m,1H), 4.49-4.63 (m, 2H), 4.89 (bs, 1H), 7.3-7.36 (m, 2H), 7.41 (t, 2H),7.59 (d, 2H), 7.77 (d, 2H).

Step 4 (9H-fluoren-9-yl)methyl (2-aminoethyl)(hexyl)carbamatehydrochloride

Acetyl chloride (2.84 mL, 40.0 mmol) was added dropwise to an icebathcooled vial of MeOH (10 mL, 247 mmol) and stirred for 5 min. Thissolution was then added to (9H-fluoren-9-yl)methyl(2-((tert-butoxycarbonyl)amino)ethyl)(hexyl)carbamate (3.7 g, 7.93 mmol)and the reaction stirred at room temperature for 2.5 h. The solvent wasconcentrated in vacuo to yield (9H-fluoren-9-yl)methyl(2-aminoethyl)(hexyl)carbamate hydrochloride (2.92 g, 91%) as a whitesolid.

LC/MS: m/z: 367.3 [M+H]⁺

1H NMR (400 MHz, DMSO-d6) δ 0.82-0.97 (m, 4H), 0.99-1.51 (m, 7H),2.68-2.88 (m, 3H), 3.23-3.39 (m, 3H), 4.23-4.4 (m, 2H), 4.52 (d, 1H),7.29-7.37 (m, 2H), 7.37-7.46 (m, 2H), 7.64 (d, 2H), 7.83-8.09 (m, 5H).

Intermediate D (S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)phenethyl)carbamate

Step 1 Methyl 4-(2-hydroxyethyl)benzoate

4-(2-Hydroxyethyl)benzoic acid (2.41 g, 14.5 mmol) was dissolved in MeOH(5 mL) and H₂SO₄ (0.06 mL, 1.13 mmol) added. The reaction was heated ina microwave at 120° C. for 1 h and was then evaporated in vacuo. Theresidue was dissolved in EtOAc (150 mL), washed with 8% NaHCO3 (aq) (150mL), brine (50 mL), dried with a phase separator and evaporated in vacuoto yield methyl 4-(2-hydroxyethyl)benzoate (2.41 g, 92%) as a pale oil.

LC/MS: m/z: 181.0 [M+H]⁺

1H NMR (500 MHz, CDCl₃) δ 2.94 (t, 2H), 3.88-3.94 (m, 5H), 7.32 (d, 2H),7.97-8.02 (m, 2H).

Step 2 Methyl 4-(2-oxoethyl)benzoate

Methyl 4-(2-hydroxyethyl)benzoate (2.41 g, 13.37 mmol) was dissolved inDCM (50 mL) and Dess-Martin Periodinane (6.24 g, 14.7 mmol) added. Thereaction was stirred at room temperature for 2 h and was then quenchedby addition of 15% Na2S2O3/NaHCO3 3:2 (aq) (50 mL) and stirredvigorously for 30 min. The phases were separated and the aqueous phaseextracted with DCM (2×50 mL). The combined organic phases were driedwith a phase separator and evaporated in vacuo. The residue was purifiedby automated flash chromatography on a Biotage® KP-SIL 100 g column. Agradient from 5-40% of EtOAc in heptane over 9 CV followed by 50% EtOAcin heptane over 3 CV were used as mobile phase. The product wascollected using the wavelength 245 nm. The product was collected andevaporated in vacuo to yield methyl 4-(2-oxoethyl)benzoate (0.880 g,36.9%) as a yellow solid.

1H NMR (500 MHz, CDCl₃) δ 3.78 (d, 2H), 3.93 (s, 3H), 7.29-7.34 (m, 2H),8.03-8.09 (m, 2H), 9.79 (t, 1H).

Step 3 (S)-Methyl4-(2-((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)ethyl)benzoate

(R)-3,5-Diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(Intermediate A, 2.00 g, 5.56 mmol), methyl 4-(2-oxoethyl)benzoate (1.09g, 6.12 mmol) and DIPEA (0.971 mL, 5.56 mmol) were dissolved in MeOH (4mL) and stirred at room temperature for 30 min. Sodium cyanoborohydride(1.05 g, 16.7 mmol) and acetic acid (0.318 mL, 5.56 mmol) were thenadded and the reaction stirred at room temperature for 3 h. The reactionwas quenched by addition of 8% NaHCO₃ (aq). The reaction was dilutedwith EtOAc (100 mL), stirred for 30 min and the phases separated. Theaqueous phase was extracted with EtOAc (100 mL). The combined organicphases were washed with brine (50 mL), dried with a phase separator andevaporated in vacuo. The residue was dissolved in DCM (20 mL) and BOC₂O(1.290 mL, 5.56 mmol) added. The mixture was stirred at room temperaturefor 5.5 h and was then concentrated in vacuo. The residue was purifiedby automated flash chromatography on a Biotage® KP-SIL 100 g column. Agradient from 10-80% of EtOAc in heptane over 10 CV was used as mobilephase. The product was collected using the wavelength 270 nm. Theproduct peak were collected and evaporated in vacuo to yield (S)-methyl4-(2-((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)ethyl)benzoate(1.84 g, 54%) as a pale solid.

LC/MS: m/z: 611.4 [M+H]⁺

¹H NMR (500 MHz, DMSO-d₆) δ 1.36 (d, 9H), 2.15-2.26 (m, 4H), 2.45-2.49(m, 2H), 2.75 (t, 2H), 2.93 (dd, 1H), 3.01-3.4 (m, 5H), 3.84 (s, 3H),6.97 (bs, 2H), 7.05-7.17 (m, 4H), 7.21 (d, 2H), 7.82 (d, 2H), 7.85-7.94(m, 1H).

Step 4(S)-4-(2-((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)ethyl)benzoicacid

(S)-Methyl4-(2-((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)ethyl)benzoate(1.84 g, 2.56 mmol) was dissolved in MeOH (20 mL) and sodium hydroxide(3.37 mL, 12.8 mmol) added. The reaction was stirred at room temperaturefor 3 days. The solvent (MeOH) was then concentrated in vacuo. Water (50mL) and MeTHF (50 mL) were added and the pH adjusted to ˜2 with 3 M HCl(aq). The phases were separated and the aqueous phase extracted withL-MeTHF (50 mL). The combined organic phases were dried with Na2SO4 (s),filtered and evaporated in vacuo to yield(S)-4-(2-((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)ethyl)benzoicacid (1.53 g, 100%) as a pale solid.

LC/MS: m/z: 597.4 [M+H]⁺

1H NMR (400 MHz, DMSO-d6) δ 1.33 (s, 9H), 2.21 (d, 4H), 2.38-2.49 (m,2H), 2.73 (t, 2H), 2.83-3.43 (m, 6H), 6.84-7.26 (m, 8H), 7.78-7.98 (m,3H), 12.80 (bs, 1H).

Step 5 (S)-tert-Butyl4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)(hexyl)amino)ethyl)carbamoyl)phenethyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

(S)-4-(2-((tert-Butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)ethyl)benzoicacid (1.24 g, 1.77 mmol),2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouroniumtetrafluoroborate (0.623 g, 1.94 mmol) and DIPEA (1.54 mL, 8.83 mmol)were dissolved in DCM (30 mL) and stirred at room temperature for 5 minbefore addition of (9H-fluoren-9-yl)methyl(2-aminoethyl)(hexyl)carbamate hydrochloride (Intermediate C, 0.95 g,2.12 mmol). The reaction was stirred for 2.5 h. The reaction was washedwith 8% NaHCO3 (aq) (150 mL). The aqueous phase was extracted with DCM(150 mL). The combined organic phases were dried with a phase separatorand evaporated in vacuo. The residue was purified by automated flashchromatography on a Biotage® KP-SIL 100 g column. A gradient from 20-80%of EtOAc in heptane over 9 CV followed by 80% EtOAc in heptane over 3 CVwere used as mobile phase. The product was collected and evaporated invacuo to yield (S)-tert-butyl4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)(hexyl)amino)ethyl)carbamoyl)phenethyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(1.56 g, 93%) as a pale solid.

LC/MS: m/z: 945.6 [M+H]⁺

¹H NMR (500 MHz, DMSO-d₆) δ 0.82 (t, 3H), 0.87-0.97 (m, 2H), 1-1.27 (m,5H), 1.33 (s, 9H), 1.38-1.47 (m, 1H), 2.15-2.27 (m, 4H), 2.45 (dd, 1H),2.69 (s, 2H), 2.81-2.89 (m, 1H), 2.9-3 (m, 1H), 3.01-3.41 (m, 11H),4.15-4.27 (m, 2H), 4.48 (d, 1H), 6.96 (bs, 2H), 7.05-7.18 (m, 6H), 7.30(t, 2H), 7.39 (t, 2H), 7.55-7.65 (m, 2H), 7.71 (dd, 2H), 7.8-7.97 (m,3H), 8.37-8.51 (m, 1H).

Step 6 (S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)phenethyl)carbamate

(S)-tert-butyl4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)(hexyl)amino)ethyl)carbamoyl)phenethyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(1.59 g, 1.68 mmol) was dissolved in THF (15 mL) and piperidine (1.67mL, 16.8 mmol) added. The reaction was stirred at room temperature for 3h and was then concentrated in vacuo. The residue was purified byautomated flash chromatography on a Biotage® KP-SIL 100 g column. Agradient from 4-8% of (2 M ammonia in MeOH) in DCM over 5 CV followed by8% of (2 M ammonia in MeOH) in DCM over 10 CV were used as mobile phase.The product was collected and evaporated in vacuo to yield(S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)phenethyl)carbamate(1.03 g, 85%) as a pale solid.

LC/MS: m/z: 723.6 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.84 (t, 3H), 1.19-1.44 (m, 17H), 2.15-2.29(m, 4H), 2.45 (dd, 1H), 2.54 (t, 2H), 2.65-2.75 (m, 4H), 2.94 (dd, 1H),3.02-3.18 (m, 2H), 3.18-3.3 (m, 3H), 3.3-3.4 (m, 4H), 6.97 (bs, 2H),7.05-7.2 (m, 7H), 7.73 (d, 2H), 7.79-7.97 (m, 1H), 8.32 (t, 1H).

Intermediate E (S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate

Step 1 (S)-methyl4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzoate

(R)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(Intermediate A, 2.17 g, 6.22 mmol), methyl 4-formylbenzoate (1.12 g,6.84 mmol) and DIPEA (1.09 mL, 6.22 mmol) were dissolved in MeOH (15 mL)and stirred at room temperature for 1 h. Sodium cyanoborohydride (0.782g, 12.4 mmol) and acetic acid (0.392 mL, 6.84 mmol) were then added andthe reaction stirred at room temperature for 3 h. When monitoring byLCMS analysis showed uncomplete conversion, methyl 4-formylbenzoate (200mg, 1.22 mmol) was added and stirring was continued for 3 h. Then againmethyl 4-formylbenzoate (200 mg, 1.22 mmol) and sodium cyanoborohydride(200 mg, 3.18 mmol) were added and stirring was continued for 18 h. Thereaction was quenched by addition of 8% NaHCO3 (aq). The solvent wasconcentrated in vacuo. The residue was dissolved in EtOAc (250 mL) and8% NaHCO₃ (aq) 250 mL), shaken and the phases separated. The aqueousphase was extracted with EtOAc (250 mL). The combined organic phaseswere dried with Na2SO4 (s), filtered and evaporated in vacuo. Theresidue was dissolved in DCM (50 mL), BOC₂O (1.63 g, 7.46 mmol) addedand the reaction stirred at room temperature for 2 h. The mixture wasconcentrated in vacuo and was then purified by automated flashchromatography on a Biotage® KP-SIL 100 g column. A gradient from 20-80%of EtOAc in heptane over 12 CV was used as mobile phase. The product wascollected using the wavelength 265 nm. The product peak was evaporatedin vacuo to yield (S)-methyl4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzoate(2.85 g, 77%) as a solid.

LC/MS: m/z: 597.5 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 1.36 (d, 9H), 2.21 (s, 3H), 2.24-2.38 (m,1H), 2.45-2.57 (m, 2H), 2.95-3.3 (m, 4H), 3.83 (s, 3H), 4.33-4.51 (m,2H), 6.97 (bs, 2H), 7.04-7.17 (m, 4H), 7.22 (d, 2H), 7.76-7.96 (m, 3H).

Step 2(S)-4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzoicacid

(S)-methyl4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzoate(2.85 g, 4.77 mmol) was dissolved MeOH (40 mL) and sodium hydroxide, 3.8M (6.28 mL, 23.9 mmol) added. The reaction was stirred at roomtemperature for 24 h and the solvent (MeOH) was then concentrated invacuo. Water (75 mL) and MeTHF (75 mL) were added and the pH adjusted to˜2 with 3 M HCl (aq). The phases were separated and the aqueous phaseextracted with MeTHF (75 mL). The combined organic phases were driedwith a phase separator and evaporated in vacuo to yield(S)-4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzoicacid (2.75 g, 99%) as a solid.

LC/MS: m/z: 583.4 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 1.32-1.42 (m, 9H), 2.20 (s, 3H), 2.24-2.38(m, 1H), 2.5-2.55 (m, 2H), 2.92-3.36 (m, 4H), 4.27-4.54 (m, 2H), 6.97(bs, 2H), 7.03-7.17 (m, 4H), 7.19 (d, 2H), 7.78-8 (m, 3H), 12.85 (bs,1H).

Step 3 (S)-tert-butyl4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)(hexyl)amino)ethyl)carbamoyl)benzyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

(S)-4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzoicacid (1.61 g, 2.76 mmol),2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouroniumtetrafluoroborate (0.975 g, 3.04 mmol) and DIPEA (2.41 mL, 13.8 mmol)were dissolved in DCM (150 mL) and stirred at room temperature for 5 minbefore addition of (9H-fluoren-9-yl)methyl(2-aminoethyl)(hexyl)carbamate hydrochloride (Intermediate C, 1.48 g,3.31 mmol). The reaction was stirred for 1.5 h. The reaction was washedwith 8% NaHCO3 (aq) (100 mL). The aqueous phase was extracted with DCM(100 mL). The combined organic phases were dried with a phase separatorand evaporated in vacuo. The residue was purified by automated flashchromatography on a Biotage® KP-SIL 100 g column. A gradient from 20-80%of EtOAc in heptane over 12 CV followed by 80% EtOAc in heptane over 3CV were used as mobile phase. The product was collected and evaporatedin vacuo to yield (S)-tert-butyl4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)(hexyl)amino)ethyl)carbamoyl)benzyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(2.38 g, 93%) as a pale solid.

LC/MS: m/z: 931.5 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.82 (t, 3H), 0.86-0.96 (m, 1H), 0.99-1.27(m, 6H), 1.27-1.48 (m, 10H), 2.19 (s, 3H), 2.23-2.35 (m, 1H), 2.45-2.54(m, 2H), 2.8-2.9 (m, 1H), 2.9-3.05 (m, 1H), 3.05-3.38 (m, 8H), 4.16-4.32(m, 3H), 4.39-4.56 (m, 2H), 6.97 (bs, 2H), 7.02-7.18 (m, 6H), 7.30 (t,2H), 7.39 (t, 2H), 7.60 (t, 2H), 7.64-7.76 (m, 2H), 7.78-7.99 (m, 3H),8.37-8.52 (m, 1H).

Step 4 (S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate

(S)-tert-butyl4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)(hexyl)amino)ethyl)carbamoyl)benzyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(2.38 g, 2.55 mmol) was dissolved in THF (25 mL) and piperidine (2.53mL, 25.6 mmol) added. The reaction was stirred at room temperature for20 h. The solvent was evaporated in vacuo. The residue was purified byautomated flash chromatography on a Biotage® KP-SIL 100 g column. Agradient from 2.5-8% of (2 M ammonia in MeOH) in DCM over 9 CV followedby 8% of (2 M ammonia in MeOH) in DCM over 3 CV were used as mobilephase. The product was collected and evaporated in vacuo to yield(S)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate(1.60 g, 88%) as a pale solid.

LC/MS: m/z: 709.5 [M+H]⁺

1H NMR (500 MHz, DMSO-d6) δ 0.84 (t, 3H), 1.17-1.31 (m, 6H), 1.31-1.45(m, 11H), 2.20 (s, 3H), 2.24-2.37 (m, 1H), 2.51-2.57 (m, 3H), 2.68 (t,2H), 2.91-3.06 (m, 1H), 3.06-3.4 (m, 6H), 4.23-4.36 (m, 1H), 4.4-4.53(m, 1H), 6.97 (bs, 2H), 7.04-7.19 (m, 6H), 7.72 (d, 2H), 7.79-7.98 (m,1H), 8.28-8.35 (m, 1H).

Intermediate F 9H-fluoren-9-yl)methyl(3-(4-formylpiperidin-1-yl)-3-oxopropyl)carbamate

Step 1 (9H-fluoren-9-yl)methyl3-(4-(hydroxymethyl)piperidin-1-yl)-3-oxopropylcarbamate

To a stirred solution of piperidin-4-ylmethanol (200 mg, 1.74 mmol),3-(((9H-fluoren-9-yl)methoxy)carbonylamino)propanoic acid (541 mg, 1.74mmol) and HATU (660 mg, 1.74 mmol) in dry DMF (5 mL) was added DIPEA(0.910 mL, 5.21 mmol). The yellow mixture was stirred at ambienttemperature over night. The reaction mixture was diluted with EtOAc (50mL) and washed with 15% aq NaCl solution (50 mL) and 30% aq NaClsolution (50 mL). Solvents were evaporated and the residue was purifiedby preparative HPLC. After freezedrying the residue was redissolved inDCM and Heptane was added. Evaporation afforded the title compound as acolourless solid (378 mg, 53%).

LC/MS: m/z: 409 [M+H]⁺

1H NMR (500 MHz, DMSO) δ 0.88-1.09 (m, 2H), 1.51-1.73 (m, 3H), 2.44 (t,2H), 2.47-2.52 (m, 1H+DMSO peak), 2.93 (t, 1H), 3.18 (dd, 2H), 3.23 (d,2H), 3.80 (d, 1H), 4.20 (t, 1H), 4.29 (d, 2H), 4.37 (d, 1H), 7.21 (t,1H), 7.33 (dt, 2H), 7.41 (t, 2H), 7.68 (d, 2H), 7.89 (d, 2H).

Step 2 (9H-fluoren-9-yl)methyl(3-(4-formylpiperidin-1-yl)-3-oxopropyl)carbamate

To a solution of (9H-fluoren-9-yl)methyl3-(4-(hydroxymethyl)piperidin-1-yl)-3-oxopropylcarbamate (362 mg, 0.89mmol) in DCM (5 mL) was added solid Dess-Martin Periodinane (413 mg,0.97 mmol). The resulting suspension was stirred at ambient temperaturefor 2.5 h. To the reaction mixture was added 10% Na₂S₂O₃ (3 mL), 10%NaHCO₃ (3 mL) and DCM (2 mL), the mixture was stirred vigorously for 10min and allowed to settle. The layers were separated and the water phasecontaining unsoluble salts was extracted repeatedly with DCM. Thecombined organic phases were dried over MgSO₄, filtered and the solventswere evaporated to leave crude product (440 mg) which was used withoutfurther purification.

LC/MS: m/z: 407 [M+H]⁺

Intermediate G (R)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate

Step 1 (R)-allyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

(3,5-diamino-6-chloropyrazin-2-yl)(1H-imidazol-1-yl)methanone, (858 mg,3.59 mmol) and (R)-allyl (3-amino-2-(2-methylbenzyl)propyl)carbamate(Intermediate A, Step 5) (990 mg, 3.77 mmol) were suspended in NMP (15mL) and heated to 100° C. for 4 h. The reaction mixture was allowed tocool to room temperature. EtOAc (50 mL) and water (50 mL) were added,shaken and the phases separated. The aqueous phase was extracted withEtOAc (2×50 mL). The combined organic phases were washed with 0.1 M HCl(aq) (50 mL), water (2×50 mL), brine (50 mL), dried with a phaseseparator and evaporated in vacuo. The residue was purified by automatedflash chromatography on a Biotage® KP-SIL 100 g column. A gradient from20-80% of EtOAc in heptane over 9 CV was used as mobile phase. Theproduct was collected using the wavelength 270 nm. The collectedfractions were pooled and evaporated in vacuo to yield (R)-allyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(714 mg, 45.9%) as a pale solid.

¹H NMR (500 MHz, DMSO-d₆) δ 1.9-2.02 (m, 1H), 2.23 (s, 3H), 2.41-2.55(m, 2H), 2.91-3.09 (m, 2H), 3.12-3.23 (m, 2H), 4.48 (d, 2H), 5.17 (d,1H), 5.24-5.32 (m, 1H), 5.85-5.96 (m, 1H), 6.97 (bs, 2H), 7.04-7.15 (m,3H), 7.21-7.25 (m, 1H), 7.28 (t, 1H), 8.00 (t, 1H).

LC/MS: m/z 433.3 [M+H]⁺

Step 2(S)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide

(R)-allyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(694 mg, 1.60 mmol), 1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (688mg, 4.41 mmol), palladium (II) acetate (14.4 mg, 0.06 mmol) andtriphenylphosphine (50.5 mg, 0.19 mmol) were dissolved in DCM (7 mL) andheated to 35° C. for 1.5 h. A suspension was formed. DCM (50 mL) and 5%Na₂CO₃ (aq) (50 ml) were added and the mixture stirred for 15 min. Thephases were separated and the aqueous phase extracted with DCM (50 mL).The combined organic phases were dried with a phase separator andevaporated in vacuo. The compound was purified by preparative HPLC on aKromasil C8 column (10 μm 250×50 ID mm) using a gradient of 5-45%acetonitrile in H₂O/MeCN/AcOH 95/5/0.2 buffer over 20 minutes with aflow of 100 mL/min. The compounds were detected by UV at 270 nm. Theproduct fractions were collected and freeze-dried to yield 586 mg of theacetic acid salt. The salt was stirred in EtOAc (50 mL) and 5% Na₂CO₃(aq) for 15 min and the phases were then separated. The aqueous phasewas extracted with EtOAc (3×50 mL). The combined organic phases weredried with Na₂SO₄ (s), filtered and evaporated in vacuo to yield(S)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(411 mg, 73.5%) as a light yellow solid. Chiral purity: 60% ee,determined with chiral SFC column: ChiralPak IC (150×4.6 mm), 3 mparticle size, mobile phase: 20% MeOH/DEA 100:0.5 in CO₂, 120 bar, flowrate 4 mL/min.

¹H NMR (500 MHz, DMSO-d₆) δ 1.73 (bs, 2H), 2.01-2.12 (m, 1H), 2.50 (s,3H), 2.64-2.73 (m, 2H), 2.76-2.86 (m, 2H), 3.4-3.53 (m, 2H), 7.19 (bs,2H), 7.28-7.41 (m, 4H), 8.39 (t, 1H).

LC/MS: m/z 349.2 [M+H]⁺

Step 3 (R)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate

(S)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(1.19 g, 3.41 mmol), BOC₂O (0.871 mL, 3.75 mmol) and DIPEA (0.596 mL,3.41 mmol) were dissolved in DCM (100 mL) and stirred at roomtemperature for 18 h. The reaction mixture was washed with 0.1 M HCl(aq), dried with a phase separator and evaporated in vacuo. The residuewas purified by automated flash chromatography on a Biotage® KP-SIL 100g column. A gradient from 30-70% of EtOAc in heptane over 9 CV followedby 70% EtOAc in heptane over 3 CV were used as mobile phase. The productwas collected using the wavelength 268 nm. The product was evaporated invacuo to yield (1.150 g, 75%) as a pale solid.

The enantiomer was purified using chiral preparative HPLC (Column:ChiralPak AY (250×20 mm), 20 m particle size, mobile phase:heptane/EtOH/TEA 20/80:0.1, flow rate 120 m/min). The first elutingcompound was collected to yield 866 mg, chiral purity: 98.6% ee. Opticalrotation [α]_(D) ²⁰=−6 (acetonitrile, c=1).

¹H NMR (500 MHz, DMSO-d₆) δ 1.38 (d, 9H), 1.88-2 (m, 1H), 2.23 (s, 3H),2.39-2.49 (m, 2H), 2.83-2.93 (m, 1H), 2.93-3.03 (m, 1H), 3.09-3.23 (m,2H), 6.89 (t, 1H), 6.97 (bs, 2H), 7.04-7.15 (m, 3H), 7.22 (d, 1H), 8.01(t, 1H).

LC/MS: m/z 449.2 [M+H]⁺

Step 4(S)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide

Acetyl chloride (1.422 mL, 20 mmol) was added dropwise to an icebathcooled flask of MeOH (5 mL, 123.59 mmol). The mixture was stirred for 5min and was then added to a flask of (R)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)carbamate(866 mg, 1.93 mmol). The reaction was stirred at room temperature for 45min and was then evaporated in vacuo. The residue was stirred in EtOAc(125 mL) and 8% NaHCO₃ (aq) (125 mL) for 15 min. The phases wereseparated and the aqueous phase extracted with EtOAc (3×125 mL). Thecombined organic phases were dried with Na₂SO₄ (s), filtered andevaporated in vacuo to yield(S)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(459 mg, 68.2%) as a pale solid. Chiral purity: 99% ee, determined withchiral SFC column: Lux C4 (150×4.6 mm), 3 m particle size, mobile phase:35% EtOH/NH₃ 100:0.5 in CO₂, 120 bar, flow rate 4 mL/min. Opticalrotation [α]_(D) ²⁰=−45.9 (CHCl₃, c=1).

¹H NMR (500 MHz, DMSO-d₆) δ 1.57 (bs, 2H), 1.78-1.88 (m, 1H), 2.26 (s,3H), 2.39-2.48 (m, 2H), 2.52-2.62 (m, 2H), 3.18-3.28 (m, 2H), 6.95 (bs,2H), 7.04-7.17 (m, 4H), 8.14 (t, 1H).

LC/MS: m/z 349.2 [M+H]⁺

Step 5 (R)-(9H-fluoren-9-yl)methyl(2-(4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzamido)ethyl)(hexyl)carbamate

(S)-3,5-diamino-N-(3-amino-2-(2-methylbenzyl)propyl)-6-chloropyrazine-2-carboxamide(215 mg, 0.62 mmol), (9H-fluoren-9-yl)methyl(2-(4-formylbenzamido)ethyl)(hexyl)carbamate (Intermediate H) (293 mg,0.59 mmol) and DIPEA (0.103 mL, 0.59 mmol) were dissolved in MeOH (5 mL)and stirred at room temperature for 1.5 h. Sodium cyanoborohydride (38.8mg, 0.62 mmol) and acetic acid (0.101 mL, 1.76 mmol) were then added andstirring continued for 3 h. The reaction was quenched by addition of 8%NaHCO₃ (aq). DCM (25 mL) and 8% NaHCO₃ (aq) (25 mL) were added, shakenand the phases separated. The aqueous phase was extracted with DCM (25mL). The combined organic phases were dried with a phase separator andevaporated in vacuo.

The residue and BOC₂O (0.150 mL, 0.65 mmol) were dissolved in DCM (5 mL)and stirred at room temperature for 18 h. The reaction mixture wasconcentrated in vacuo. The product was purified by automated flashchromatography on a Biotage® KP-SIL 50 g column. A gradient from 50-100%of EtOAc in heptane over 9 CV was used as mobile phase. The product wascollected using the wavelength 265 nm. The product fractions wereevaporated in vacuo to yield (R)-(9H-fluoren-9-yl)methyl(2-(4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzamido)ethyl)(hexyl)carbamate(511 mg, 93%) as a colorless film.

¹H NMR (500 MHz, DMSO-d₆) δ 0.82 (t, 3H), 0.87-0.96 (m, 1H), 0.99-1.26(m, 6H), 1.27-1.5 (m, 10H), 2.19 (s, 3H), 2.23-2.35 (m, 1H), 2.8-2.9 (m,1H), 2.9-3.04 (m, 1H), 3.05-3.39 (m, 10H), 4.16-4.32 (m, 3H), 4.39-4.57(m, 2H), 6.88-7.18 (m, 8H), 7.30 (t, 2H), 7.39 (t, 2H), 7.55-7.76 (m,4H), 7.77-7.99 (m, 3H), 8.37-8.52 (m, 1H).

LC/MS: m/z 931.4 [M+H]⁺

Step 6 (R)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate

(R)-(9H-fluoren-9-yl)methyl(2-(4-(((tert-butoxycarbonyl)(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)amino)methyl)benzamido)ethyl)(hexyl)carbamate (511 mg, 0.55 mmol) was dissolved in THF (5 mL) andpiperidine (0.543 mL, 5.49 mmol) added. The reaction was stirred at roomtemperature for 18 h. The solvent was evaporated in vacuo. The residuewas purified by automated flash chromatography on a Biotage® KP-SIL 50 gcolumn. A gradient from 2.5-8% of (2 M ammonia in MeOH) in DCM over 9 CVfollowed by 8% of (2 M ammonia in MeOH) in DCM over 3 CV were used asmobile phase. The product was collected and evaporated in vacuo to yield(R)-tert-butyl(3-(3,5-diamino-6-chloropyrazine-2-carboxamido)-2-(2-methylbenzyl)propyl)(4-((2-(hexylamino)ethyl)carbamoyl)benzyl)carbamate(326 mg, 84%) as a pale solid.

¹H NMR (500 MHz, DMSO-d₆) δ 0.84 (t, 3H), 1.12-1.45 (m, 17H), 2.20 (s,3H), 2.24-2.36 (m, 1H), 2.51-2.58 (m, 2H), 2.69 (t, 2H), 2.92-3.05 (m,1H), 3.05-3.4 (m, 7H), 4.22-4.37 (m, 1H), 4.38-4.54 (m, 1H), 6.97 (bs,2H), 7.03-7.18 (m, 6H), 7.72 (d, 2H), 7.79-7.98 (m, 1H), 8.28-8.35 (m,1H).

LC/MS: m/z 709.4 [M+H]⁺

Intermediate H (9H-fluoren-9-yl)methyl(2-(4-formylbenzamido)ethyl)(hexyl)carbamate

4-formylbenzoic acid (0.81 g, 5.40 mmol) and 4-methylmorpholine (1.424mL, 12.95 mmol) were dissolved in THF (10 mL) and cooled with anicebath. Isobutyl carbonochloridate (0.700 mL, 5.40 mmol) was addeddropwise, the mixture stirred for 30 min and (9H-fluoren-9-yl)methyl(2-aminoethyl)(hexyl)carbamate hydrochloride (Intermediate C) (1.739 g,4.32 mmol) added. The reaction was stirred at room temperature for 2.5h. The resulting suspension was extracted between EtOAc (50 mL) andwater (50 mL). The organic phase was washed with 5% citric acid (2×50mL), 10% Na₂CO₃ (aq) (50 mL), brine (50 mL), dried with Na₂SO₄ (s),filtered and evaporated in vacuo. The residue was purified by automatedflash chromatography on a Biotage® KP-SIL 100 g column. A gradient from20-80% of EtOAc in heptane over 9 CV followed by 80% EtOAc in heptaneover 3 CV were used as mobile phase. The product was collected using thewavelength 260 nm. The product peaks were evaporated in vacuo to yield(9H-fluoren-9-yl)methyl (2-(4-formylbenzamido)ethyl)(hexyl)carbamate(1.690 g, 79%) as a white solid.

1H NMR (500 MHz, CDCl₃) δ 0.82-0.95 (m, 3H), 1.08-1.46 (m, 8H),3.02-3.21 (m, 3H), 3.48-3.64 (m, 3H), 4.16-4.25 (m, 1H), 4.51-4.74 (m,2H), 7.2-7.42 (m, 5H), 7.5-7.67 (m, 3H), 7.75 (d, 2H), 7.93 (q, 3H),10.06 (s, 1H).

LC/MS: m/z 499.5 [M+H]⁺

Intermediate I4-formyl-N-(2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)benzamide

Step 1 tert-butyl 2-(hexylamino)ethylcarbamate

To a 10 L flange flask was charged hexylamine (1020 mL, 7.76 mol),Na₂CO₃ (206 g, 1.94 mol) and MeCN (2560 mL). The mixture was heated to70° C. over 1 h. To the mixture was charged a solution of tert-butyl2-bromoethylcarbamate (371.3 g, 289.6 g active, 1.29 mol) in MeCN (740ml) slowly, over 1½ h. The reaction was stirred at 70° C. overnight. Thereaction was cooled to 50° C. and concentrated in vacuo. To the stirredresidue was charged water (2100 mL) and EtOAc (4200 ml). The layers wereseparated and the organic phases were washed with water (3×2100 ml). Theorganic phases were concentrated in vacuo and azeotroped sequentiallywith water (3×870 ml) and EtOH (3×870 ml) to provide 368.6 g (315.8 gactive, quantitative yield) of stage 1 as a clear oil.

1H-NMR (270 MHz, CDCl₃,) δ 4.95 (1H, br s), 3.29-3.19 (m, 2H), 2.71 (t,2H), 2.58 (t, 2H), 1.50-1.40 (br. m, 11H), 1.35-1.23 (m, 6H), 0.89 (t,3H).

LC/MS: m/z 245 [M+H]⁺

Step 2 tert-butyl2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethylcarbamate

To a 10 L flange flask was charged D-xylose (124.3 g, 0.83 mol),tert-butyl 2-(hexylamino)ethylcarbamate (step 1, 181.8 g, 155.8 gactive, 0.49 mol), EtOH (5000 ml) and DIPEA (110.8 ml, 0.64 mol). Themixture was heated to 35° C. for 1 h, until dissolved. To the solutionwas charged AcOH (36.5 mL, 0.64 mol) and the resulting mixture stirredat 35° C. for 15 min. To this was charged NaCNBH₃ (56.1 g, 0.89 mol),portionwise over 5 min at 35° C. No exotherm or gas evolution noted. Thereaction was stirred at 35° C. overnight. To this was charged NaCNBH₃(8.0 g, 0.13 mol) and stirred at 35° C. for 3 h. The reaction wasallowed to cool to room temperature overnight. To the mixture wascharged sat. aq. NaHCO₃ (1600 mL) over 10 min, a slightly exothermicreaction was observed. The mixture was stirred at room temperature overthe weekend. The EtOH was removed in vacuo and combined with a secondbatch of the same scale (181.8 g stage 1) for work-up. To the mixturewas charged NaCl (311 g). The organic phases were extracted with 10%MeOH/DCM (3×3000 ml) and the combined organic phases were concentratedin vacuo. The resulting residue was purified via chromatography (SiO₂, 7kg), packed, loaded and eluted with 5% MeOH/DCM+0.2% 7 N NH₃ in MeOH (35L), 50% MeOH/DCM+0.2% 7 N NH₃ in MeOH (75 L), then flushing with 65%MeOH/DCM+0.2% 7 N NH₃ in MeOH (20 L). Product containing fractions werecombined, evaporated in vacuo and azeotroped with MeOH (2×500 mL) toprovide 387 g (366 g active, 76%) of the subtitle compound as a yellowgum.

1H-NMR (270 MHz, DMSO-d6) δ 6.80 (br.s, 1H), 4.80-4.30 (br. m, 3H),4.22-4.05 (br. m, 1H), 3.74-3.60 (br. m, 1H), 3.59-3.48 (m, 1H),3.47-3.20 (m, 6H), 3.08-2.87 (br. m, 2H), 2.70-2.45 (br. m, 3H),1.51-1.35 (m, 10H), 1.32-1.16 (m, 7H), 0.85 (t, 3H).

LC/MS: m/z 401 [M+H]⁺

Step 3 (2R,3R,4 S)-5-((2-aminoethyl)(hexyl)amino)pentane-1,2,3,4-tetraol

To a solution of tert-butyl2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethylcarbamate(step 2, 365 g, 345 g active, 0.91 mol) in MeOH (520 ml) at roomtemperature was charged 4 M HCl in dioxane (1710 ml, 6.84 mol) over 30min, maintaining a temperature <31° C. The reaction was stirred at roomtemperature for 1.5 h. The reaction mixture was concentrated in vacuo toprovide 485.1 g (306 g active, 95%) of the subtitle compound as a purpleblack tar.

1H-NMR (270 MHz, DMSO-d6) δ 8.41 (br. s, 3H), 4.20-3.00 (br. m, 16H),1.77-1.62 (br. m, 2H), 1.37-1.17 (br. m, 6H), 0.87 (br. t, 3H).

MS ES: m/z 279 [M+H]⁺

Step 44-formyl-N-(2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)benzamide

To a 5 L flange flask was charged 4-formylbenzoic acid (107.6 g, 0.72mol) and DMF (1400 ml) at room temperature. To this was charged4-methylmorpholine (255.5 ml, 2.3 mol). The mixture was cooled to −15°C. over 5 min. To this was charged isobutyl chloroformate (93.4 ml, 0.71mol) over 15 min, maintaining a reaction temperature <−10° C. Thereaction was stirred at −10° C. to −15° C. for 1 h. In a separate flask,(2R,3R,4S)-5-((2-aminoethyl)(hexyl)amino)pentane-1,2,3,4-tetraol (step3, 383.9 g, 245.7 g active, 0.71 mol) was dissolved in DMF (1400 ml) and4-methylmorpholine (123 ml, 1.1 mol) at 45° C. over 1 h. To the mixedanhydride mixture was charged the step 3 containing mixture over 15 min,maintaining a reaction temperature <−5° C. The resulting mixture wasallowed to warm to room temperature overnight. To the reaction mixturewas charged sat. aq. NaHCO₃ (2500 ml) slowly, over 30 min. The resultingmixture was concentrated in vacuo. To the residue was charged sat. aq.NaHCO₃ (1200 ml) and stirred with DCM (4900 ml) for 10 min at RT. Thelayer were separated and the aqueous was extracted with 13% MeOH/DCM(2870 ml). The combined organic phases were washed with sat. aq. NaHCO₃(1200 ml) and concentrated in vacuo to provide 395.3 g. The residue wascombined with 10 g crude material recovered from an intermediate scalereaction and purified via chromatography [SiO₂, 3 kg; loaded and packedwith 5% MeOH/DCM; eluted with 5% MeOH/DCM (30 L), 10% MeOH/DCM (20 L),20% MeOH/DCM (10 L), 30% MeOH/DCM (30 L), 40% MeOH/DCM (20 L) and 100%MeOH (80 L)]. Product fractions were combined (100 L) and concentratedin vacuo to provide 138.4 g (112 g active, 39% yield) of the subtitlecompound. ¹H NMR indicated 81.4 w/w % activity with ca. 5.5% remainingformylbenzamide impurity. HPLC-MS analysis indicated 97.4% purity. Mixedfractions (20 L) were combined and concentrated in vacuo to provide 46 gof material with a purity of 74.5% by HPLC. This was re-purified viachromatography (SiO₂, 600 g), eluting with 10%-30% MeOH/DCM (18 L).Product fractions were combined and concentrated in vacuo to provideadditional 21.0 g (20.3 g active, 44%) of the subtitle compound.

1H-NMR (270 MHz, DMSO-d6): δ 10.08 (s, 1H), 8.60 (t, 1H), 8.02 (d, 2H),7.97 (d, 2H), 4.70-4.20 (br. m, 4H), 3.66-3.52 (m, 2H), 3.46-3.34 (m,8H), 2.65-2.58 (m, 2H), 2.51-2.44 (m, 2H), 1.40-1.28 (br. m, 2H),1.19-1.08 (m, 6H), 0.79 (t, 3H).

LC/MS: m/z 411 [M+H]⁺

Enzymatic desymmetrisation of 2-(2-methylbenzyl)propane-1,3-diamine

The enzymatic desymmetrisation of 2-(2-methylbenzyl)propane-1,3-diaminewas investigated using diallylcarbonate and a range of immobilizedlipases.

Immobilised Lipase Screen:

To a solution of 2-(2-methylbenzyl)propane-1,3-diamine (0.60 g, 3.37mmol) in 1,4-dioxan (30 mL) was added diallylcarbonate (0.507 mL, 3.53mmol). 1.0 mL aliquots of this solution were added to 10 mL screw captest tubes containing the enzymes (0.02 g) described in Table 1. Thetubes were shaken at 500 rpm/30° C. for 3 days. Each tube was sampledand analysed by Reverse Phase HPLC (4.6×50 mm Thermoquest Hypercarb, UVdetection at 260 nm) and Chiral HPLC (4.6×250 mm Chiralpak IC3, UVdetection at 260 nm).

Six of the screened lipases showed more than 20% conversion after 3 days(Table 1).

TABLE 1

Enzyme Conversion¹ R/S ratio² IMM CALB (Candida Antartica Lipase B) 33.20.87 IMM CALBY (Candida Antartica Lipase B) 52.8 0.81 Novozym 435(Candida Antarctica Lipase B) 75.8 1.76 Amano Lipas PS-C1 (PseudomonasCepacia Lipase) 31.4 9.8  Amano Lipase PS-IM (Pseudomonas CepaciaLipase) 21.2 5.0  Amano Lipase PS-D (Pseudomonas Cepacia Lipase) 33.412.3  No enzyme (Control) — — ¹Conversion to mono-allyl carbamate areindicated in % area from HPLC. ²R/S ratios are indicated in % area Renantiomer divided with % area S enantiomer from chiral HPLC.

Solvent Screen with Amano Lipase PS-IM:

A solvent screen with Amano Lipase PS-IM was carried out in 20 relativevolumes of the solvents with 1.1 equivalents diallylcarbonate, 1 massequivalent Amano PS-IM and 0.1 g of2-(2-methylbenzyl)propane-1,3-diamine. After 6 days at 30° C. theexperiments were sampled using the same methods as described for thelipase screen (Table 2). 2-(2-Methylbenzyl)propane-1,3-diamine was notvery soluble in non-polar solvents, such as methyl t-butyl ether,heptanes and cyclohexane. Addition of THF to each of the solvents did,however, allow the diamine to dissolve and the above 1:1 solventmixtures were tested. The results suggested that 2-MeTHF gave the bestenantioselectivity, despite some background reaction. In order tominimize the background carboxylation in 2-MeTHF, a gram scaleexperiment was carried out at 30° C. with excess of Amano lipase PS-IM(3 mass equivalents immobilized lipase, 1.2 equivalentsdiallylcarbonate, 20 vol 2-MeTHF). The reaction proceeded to completionwithin 3 days and after workup, crude (R)-monoallylcarbamate 1-(R) wasisolated with 85% ee. Converting the crude product into thecorresponding D-tartarate salt in ethanol increased the enantiomericexcess to 91.0% (58% overall yield from the diamine).

TABLE 2 Solvent Conversion¹ R/S ratio² TBME:THF (1:1) 72.8 6.25Heptane:THF (1:1) 88.4 5.45 Cyclohexane:THF (1:1) 89.8 6.81 Toluene 52.56.63 THF 36.8 4.91 MeTHF 86.2 7.06 MeTHF (No enzyme) 24.8 — ¹Conversionto mono-allyl carbamate are indicated in % area from HPLC. ²R/S ratiosare indicated in % area R enantiomer divided with % area S enantiomerfrom chiral HPLC.Sodium Channel ENaC Ussing Chamber Test

Human primary bronchial epithelial cells differentiated at air-liquidinterface (ALI) on snapwell permeable supports (MatTek Corporation, MA,USA, cat no AIR-100-SNP) were maintained at ALI culture in a humidifiedincubator at 37° C. and 5% CO₂. Basolateral medium (MatTek Corporation,MA, USA cat no AIR-100-MM-ASY) was changed every second day. Mucus orliquid formed on the apical side was gently aspirated every second dayto maintain viability and performance of the cells.

The transepithelial voltage between the luminal/apical and basolateralmembrane were measured in the ussing chamber consisted of vertical,in-house manufactured ussing chambers, DVC-1000 V/C clamps withpreamplifiers (World Precision Instruments), electrode kits (EK1, WorldPrecision Instruments), Power Lab with Chart5 software, water bath withexternal circulation and carbogen gas distribution and regulationsystem. Electrodes used in the set up were casted manually using 4%agarose in 0.9% sterile NaCl according to the manufacturer'srecommendation.

The ussing chambers were assembled with an empty snapwell membrane(Costar 3407) and electrodes contained in Kreb's buffer (SIGMA-ALDRICHcat no K3753) for one hour at 37° C. in presence of 95% oxygen and 5%carbon dioxide. After this equilibration step the system was compensatedfor fluid resistance and input offset.

Prior to experiment the ALI cultures were assessed for Trans EpithelialElectric Resistance (TEER) with the EndOhm device (World PrecisionInstruments), cells exhibiting a resistance between 400-600 Ohms wereused.

The equilibrated chambers were reassembled with a snapwell containingALI cells and after 20 minutes stabilisation cumulative doses of thecompounds were added to the apical side of the membrane. After the finaldose 10 μM Benzamil was added to reach maximum.

All test compounds, including the reference compound, were diluted from10 mM stocks in 100% DMSO and added to opaque 96-well plates (Greiner,Cat No. 651201). Dilution series of 12 doses were prepared in DMSO,starting with 3 μM followed by 10 times dilution steps.

Chart5 for Windows software was used to measure the value of the shortcircuit current after each compound addition. The data was transferredinto Excel and analysed using IDBS XLfit® 5.2 (ID Business SolutionsLtd). The molar concentration of test or reference compound producing50% inhibition (curve IC50) was derived by fitting data to a 4-parameterlogistic function of the form:y=(A+((B−A)/(1+(((10^C)/x)^D))))

A The bottom plateau of the curve, i.e. the final minimum y value

B The top of the plateau of the curve i.e. the final maximum y value

C The log₁₀x value at a y value of 50%.

D The Hill slope factor.

x The known x values.

Table 1 shows the pIC₅₀ values for Examples 1 to 14.

TABLE 1 Example No. pIC₅₀ 1 9.5 2 8.6 3 10.1 4 9.2 5 9.0 6 9.5 7 8.9 89.5 9 8.6 10 8.6 11 8.8 12 9.6 13 10.1 14 7.5Estimated Intestinal Mucosal Permeation Using Caco-2 Cell Monolayer

Caco-2 cells are grown in Dulbecco's Modified Eagle's medium (DMEM) on96-well transwell polycarbonate membrane inserts for 15 days untilconfluence and differentiation is reached. After integrity assessment bymeasurement of electrical resistance across the monolayer cells arewashed two times with HBSS (25 mM HEPES, pH 7.4) and incubated at 37° C.for 30 min.

Stock solutions of test compounds in DMSO are diluted with HBSS(containing 100 μM Lucifer Yellow) to reach the final concentration of10 μM; all incubations are performed in duplicate. To determine the rateof drug transport in apical to basolateral direction (A to B) 100 μL oftest compound solutions are added to the apical compartment of theTranswell insert. The basolateral compartment is filled with 300 μL ofHBSS.

For determination of basolateral to apical transport (B to A) thebasolateral compartment is filled with 300 μL of test compound solutionand 100 μL of HBSS are added to the apical compartement.

After incubation of the plate at 37° C. for 2 h 50 μL from eachcompartment are removed and transferred to 96 well plates formeasurement of fluorescence in a suitable plate reader at 485 nmexcitation and 530 nm emission to monitor monolayer integrity. For thetest compound permeability analysis 3 volumes of cold methanol are addedto each buffer sample and the samples are centrifuged at 4 C for 15minutes. An aliquot of 5 μL of the supernatant is used for LC/MS/MSanalysis.

The apparent permeability (Papp, in units of cm/s) can be calculatedusing the following equation

$P_{app} = {\frac{V_{A}}{{Area} \times {time}} \times \frac{\lbrack{drug}\rbrack_{acceptor}}{\lbrack{drug}\rbrack_{{initial},\;{donor}}}}$where V_(A) is the volume (in mL) in the acceptor well (0.3 mL for apialto basolateral), Area is the surface area of the membrane (0.143 cm2 forHTS Transwell-96 Well Permeable Supports) and time is total transporttime in s.

The apparent permeability in the apical to basolateral direction (MedianA to B Papp, 1E-6.cm/s) was measured for each of Examples 1 to 16. Noapparent permeability could be detected for any of the Examples in theapical to basolateral direction. Compounds which exhibit lowpermeability may be more desirable for delivery of ENaC inhibitors viainhaled administration.

The above description of illustrative embodiments is intended only toacquaint others skilled in the art with Applicant's invention, itsprinciples, and its practical application so that others skilled in theart may readily adapt and apply the invention in its numerous forms, asthey may be best suited to the requirements of a particular use. Thisdescription and its specific examples, while indicating embodiments ofthis invention, are intended for purposes of illustration only. Thisinvention, therefore, is not limited to the illustrative embodimentsdescribed in this specification, and may be variously modified. Inaddition, it is to be appreciated that various features of the inventionthat are, for clarity reasons, described in the context of separateembodiments, also may be combined to form a single embodiment.Conversely, various features of the invention that are, for brevityreasons, described in the context of a single embodiment, also may becombined to form sub-combinations thereof.

The invention claimed is:
 1. A compound which is:3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition, wherein the composition comprises the compound according toclaim 1, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable adjuvant, diluent or carrier.
 3. A compoundwhich is:3,5-diamino-6-chloro-N—((R)-3-((4-((2-(hexyl((2S,3R,4R)-2,3,4,5-tetrahydroxypentyl)amino)ethyl)carbamoyl)benzyl)amino)-2-(2-methylbenzyl)propyl)pyrazine-2-carboxamide:


4. A pharmaceutical composition, wherein the composition comprises thecompound according to claim 3, and a pharmaceutically acceptableadjuvant, diluent or carrier.